Preparation of phosphorothioate oligomers

ABSTRACT

Methods and intermediates for the preparation of oligomers containing diastereomerically enriched phosphorothioate linkages are disclosed.

This is a continuation of application Ser. No. 08/546,198, filed Oct.20, 1995, now U.S. Pat. No. 5,734,041, the disclosure of which is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods for the preparation ofdiastereomerically enriched phosphorothioate linked oligonucleotides,and to intermediates useful in their preparation. This invention alsorelates to sequence-specific phosphorothioate oligonucleotides havingchiral phosphorus linkages and to a novel chemical synthesis of theseand other oligonucleotides.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic of otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. Classical therapeutics hasgenerally focused upon interactions with such proteins in efforts tomoderate their disease-causing or disease-potentiating functions. Innewer therapeutic approaches, modulation of the actual production ofsuch proteins is desired. By interfering with the production ofproteins, the maximum therapeutic effect might be obtained with minimalside effects. It is the general object of such therapeutic approaches tointerfere with or otherwise modulate gene expression which would lead toundesired protein formation.

One method for inhibiting specific gene expression is with the use ofoligonucleotides. Oligonucleotides complementary to a specific targetmessenger RNA (mRNA) sequence are used. Several oligonucleotides arecurrently undergoing clinical trials for such use.

Transcription factors interact with double-stranded DNA duringregulation of transcription. Oligonucleotides can serve as competitiveinhibitors of transcription factors to modulate the action oftranscription factors. Several recent reports describe such interactions(see, Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et al.,Gene 1990, 89, 203-209.)

Oligonucleotides also have found use in diagnostic tests. Suchdiagnostic tests can be performed using biological fluids, tissues,intact cells or isolated cellular components. As with the above geneexpression inhibition, diagnostic use can take advantage of anoligonucleotide's ability to hybridize with a complementary strand ofnucleic acid. Hybridization is the sequence specific hydrogen bonding ofoligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA orDNA. The bases of such base pairs are said to be complementary to oneanother.

Oligonucleotides are also widely used as research reagents. They areuseful for understanding the function of many other biological moleculesas well as in the preparation of such other biological molecules. Oneparticular use, the use of oligonucleotides as primers in the reactionsassociated with polymerase chain reaction (PCR), has been thecornerstone for the establishment of an ever expanding commercialbusiness. The use of such PCR reactions has seemingly "exploded" as moreand more use of this very important biological tool is made. The uses ofPCR have extended into many areas in addition to those contemplated byits Nobel laureate inventor. Examples of such new areas includeforensics, paleontology, evolutionary studies and genetic counseling toname just a few. Primers are needed for each of these uses.Oligonucleotides, both natural and synthetic, serve as the primers.

Oligonucleotides also are used in other laboratory procedures. A numberof these uses are described in common laboratory manuals such asMolecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al.,Eds., Cold Spring Harbor Laboratory Press, 1989; and Current ProtocolsIn Molecular Biology, F. M. Ausubel, et. al., Eds., CurrentPublications, 1993. Such uses include Synthetic Oligonucleotide probes,Screening Expression Libraries with Antibodies and Oligonucleotides, DNASequencing, In Vitro Amplification of DNA by the Polymerase ChainReaction and Site-directed Mutagenesis of Cloned DNA from Book 2 ofMolecular Cloning, A Laboratory Manual, ibid. and DNA-ProteinInteractions and The Polymerase Chain Reaction from Vol. 2 of CurrentProtocols In Molecular Biology, ibid..

To supply the users of oligonucleotides, many scientific journals nowcontain advertisements for either oligonucleotide precursors or forcustom-synthesized oligonucleotides. This has become an importantcommercial use of oligonucleotides. Oligonucleotides can be synthesizedto have properties that are tailored for the desired use. Thus, a numberof chemical modifications have been introduced into oligonucleotides toincrease their usefulness in diagnostics, as research reagents, and astherapeutic entities. These modifications are designed, for example, toincrease binding to a target nucleic acid strand, to assist inidentification of the oligonucleotide or an oligonucleotide-targetcomplex, to increase cell penetration, to provide stability againstnucleases and other enzymes that degrade or interfere with the structureor activity of the oligonucleotides, to provide a mode of disruption(terminating event) once sequence-specifically bound to a target, or toimprove the pharmacokinetic properties of the oligonucleotides.

Since they exist as diastereomers, phosphorothioate, methylphosphonate,phosphotriester, phosphoramidate and other phosphorus oligonucleotidessynthesized using known, automated techniques result in mixtures of Rpand Sp diastereomers at the individual phosphorothioate,methylphosphonate, phosphotriester, phosphoramidate or other phosphoruslinkages. Thus, a 15-mer oligonucleotide containing 14 asymmetriclinkages has 2¹⁴, e.e. 16,384, possible stereoisomers. It is possiblethat oligomers having diastereomerically enriched linkages could possessadvantages in hybridizing to a target mRNA or DNA. Accordingly, there isa need for such oligomers.

Miller, P. S., McParland, K. B., Jayaraman, K., and Ts'o, P.O.P (1981),Biochemistry, 20:1874, found that small di-, tri- andtetramethylphosphonate and phosphotriester oligonucleotides hybridize tounmodified strands with greater affinity than natural phosphodiesteroligonucleotides. Similar increased hybridization was noted for smallphosphotriester and phosphoramidate oligonucleotides; Koole, L. H., vanGenderen, M. H. P., Reiners, R. G., and Buck, H. M. (1987), Proc. K.Ned. Adad. Wet., 90:41; Letsinger, R. L., Bach, S. A., and Eadie, J. S.(1986), Nucleic Acids Res., 14:3487; and Jager, A., Levy, M. J., andHecht, S. M. (1988), Biochemistry, 27:7237. The effects of thediastereomers of undefined streochemistry on hybridization becomes evenmore complex as chain length increases.

Bryant, F. R. and Benkovic, S. J. (1979), Biochemistry, 18:2825 studiedthe effects of diesterase on the diastereomers of ATP. Published patentapplication PCT/US88/03634 discloses dimers and trimers of 2', 5'-linkeddiastereomeric adenosine units. Niewiarowski, W., Lesnikowski, Z. J.,Wilk, A., Guga, P., Okruszek, A., Uznanski, B., and Stec, W. (1987),Acta Biochimica Polonia, 34:217, synthesized dimers of thymidine havinghigh diastereomeric excess, as did Fujii, M., Ozaki, K., Sekine, M., andHata, T. (1987), Tetrahedron, 43:3395.

Stec, W. J., Zon, G., and Uznanski, B. (1985), J. Chromatography,326:263, have reported the synthesis of certain mixtures ofphosphorothioates or methyphosphonate oligonucleotides and haveseparated them by chromatography. However, they were only able toseparate the diastereomers of certain small oligomers having a limitednumber of diastereomerically pure phosphorus linkages.

In a preliminary report, J. W. Stec, Oligonucleotides as antisenseinhibitors of gene expression: Therapeutic implications, meetingabstracts, June 18-21, 1989, noted that a non-sequence-specificthymidine homopolymer octamer - - - i.e. a (dT)₈ -mer, having"al-except-one" Rp configuration methylphosphonate linkages - - - formeda thermodynamically more stable hybrid with a 15-mer deoxyadenosinehomopolymer - - - i.e. a d(A)₁₅ -mer - - - than did a similar thymidinehomopolymer having "all-except-one" Sp configuration methylphosphonatelinkages. The hybrid between the "all-except-one" Rp (dT)₈ -mer and thed(A)₁₅ -mer had a Tm of 38° C. while the Tm of the "all-except-one" Sp(dT)₈ -mer and the d(A)₁₅ -mer was <0° C. The hybrid between a (dT)₈-mer having natural phosphodiester linkages, i.e. octathymidylic acid,and the d(A)₁₅ -mer was reported to have a Tm of 14° C. The"all-except-one" thymidine homopolymer octamers were formed from twothymidine methylphosphonate tetrameric units with high diastereomericexcess linked by a natural phosphodiester linkage.

Six or more nucleotides units are generally necessary for anoligonucleotide to be of optimal use in applications involvinghybridization. It is often preferred to have even more nucleoside unitsfor best performance, aften as many as 10 to 30. Because it has not beenpossible to stereochemically resolve more than two or three adjacentphosphorus linkages, the effects of induced chirality in the phosphoruslinkages of chemically synthesized oligonucleotides has not been wellassessed heretofore. This is because with few limited exceptions, thesequence-specific phosphorothioate, methylphosphonate, phosphotriesteror phosphoramidate oligonucleotides obtained utilizing known automatedsynthetic techniques have been mixtures with no diastereomeric excess.

Some aspects of the use of enzymatic methods to synthesizeoligonucleotides having chiral phosphorus linkages have beeninvestigated. Burgers, P. M. J. and Eckstein, F. (1979), J. BiologicalChemistry, 254:6889; and Gupta, A., DeBrosse, C., and Benkovic, S. J.(1982) J. Bio. Chem., 256:7689 enzymatically synthesizeddiastereomerically pure polydeoxy-adenylic acid having phosphorothioatelinkages. Brody, R. S. and Frey, P. S. (1981), Biochemistry, 20:1245;Eckstein, F. and Jovin, T. M. (1983), Biochemistry, 2:4546; Brody, R.S., Adler, S., Modrich, P., Stec, W. J., Leznikowski, Z. J., and Frey,P. A. (1982) Biochemistry, 21: 2570-2572; and Romaniuk, P. J. andEckstein, F. (1982) J. Biol. Chem., 257:7684-7688 all enzymaticallysynthesized poly TpA and poly ApT phosphorothioates while Burgers, P. M.J. and Eckstein, F. (1978) Proc. Natl. Acad. Sci. USA, 75: 4798-4800enzymatically synthesized poly UpA phosphorothioates. Cruse, W. B. T.,Salisbury, T., Brown, T., Cosstick, R. Eckstein, F., and Kennard, O.(1986), J. Mol. Biol., 192:891, linked three diastereomeric Rp GpCphosphorothioate dimers via natural phosphodiester bonds into a hexamer.Most recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R., andWatanabe, K. (1991) Nucleic Acids Research, 19:547, enzymaticallysynthesized RNA's having from several hundred to ten thousandnucleotides incorporating Rp linkages of high diastereomeric excess.Enzymatic synthesis, however, is disadvantageous in that it depends onsuitable polymerases that may or may not be available, especially formodified nucleoside precursors.

As reviewed by W. J. Stec and A. Wiek (1994), Angew. Chem. Int. Ed.English 33:709, the oxathiaphospholane method has been successful forthe preparation of phosphorothioates with defined stereochemistry.However, it suffers from disadvantages, such as the non-trivialpreparation of diastereomerically pure oxathiaphospholane, and thedifficulty in synthesizing and isolating satisfactorily pure oligomerslonger than 12-mers.

It would therefore be of great advantage to provide oligonucleotideshaving phosphorus linkages with controlled stereochemistry.

OBJECTS OF THE INVENTION

It is one object of this invention to provide sequence-specificoligonucleotides having chirally pure phosphorothioate linkages withhigh diastereomeric excess.

Another object is to provide phosphorus-linked oligonucleotides havingsubstantially all Rp or all Sp linkages.

A further object is to provide research and diagnostic materials forassaying bodily states in animals, especially diseased states.

It is yet another object to provide new methods for synthesizingsequence-specific oligonucleotides having chirally pure phosphorothioatelinkages, and useful intermediates therefor.

SUMMARY OF THE INVENTION

The present invention provides stereoselective methods for preparingsequence-specific oligonucleotides having chiral phosphorus linkages. Incertain preferred embodiments, these methods comprise the steps of:

reacting a first synthon of Formula I: ##STR1## wherein: Q isindependently O or S;

R¹ is a hydroxyl protecting group;

R² is a chiral auxiliary of formula --C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ;

R³ is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl,trihalomethyl, --CH₂ Si(R⁴)₃, or --CH₂ --SO_(k) R⁴ where k is 0, 1 or 2;

R⁴ is independently alkyl, aryl, aralkyl or alkaryl having up to 15carbon atoms;

R⁵ is H, --CN, --Si(R⁴)₃, SO_(k) R⁴ or halogen;

or R⁸ and R¹⁶ are each H, and R³ and R⁵, together, form one of thestructures: ##STR2## wherein: R¹⁰ and R¹¹ are H, alkyl having from 1 toabout 10 carbons, --CH₂ C(═O)OR²², --CH₂ CN, --CH₂ Si(CH₃)₃, or o- orp--C₆ H₄ --R²¹ ;

R²¹ is hydrogen, --O--C(═O)CH₃, alkoxy having from 1 to about 10carbons, --NO₂, or --N(R²²)₂ ;

R²² is independently H or alkyl having from one to about 10 carbonatoms;

p is 1 or 2;

Z¹ and Z² are independently halogen, --CN, --Si(CH₃)₃, and --C(═O)OR²² ;

R³⁰ is hydrogen, --O--C(═O)CH₃, alkoxy having from 1 to about 10carbons, or --O--Si(R₄)₃ ;

R⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

or R⁵ and R⁶, together with the atoms to which they are attached, form a5 or 6 membered ring;

R⁷ is alkyl or aralkyl having up to 15 carbon atoms;

or R⁶ and R⁷, together, form one of the structures ##STR3## wherein V,T, and Z are independently CH or N; R⁸ is H or methyl;

R¹⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

B is a nucleobase; and

n is an integer from 0 to 50;

with a second synthon of Formula II: ##STR4## wherein: R⁹ is a hydroxylprotecting group or a linker connected to a solid support; and

m is an integer from 0 to 50;

for a time and under reaction conditions effective to form a thirdsynthon of Formula III: ##STR5## contacting said third synthon with asulfurizing agent to form an oligomer of Formula IV: ##STR6## wherein Dis said phosphorothioate linkage having the formula: ##STR7##

In preferred embodiments, said phosphorothioate linkage isdiastereomerically enriched. In other preferred embodiments about 75% ofthe phosphorothioate linkage is in a single stereoisomeric form. Infurther preferred embodiments about 85% of the phosphorothioate linkageis in a single stereoisomeric form. In especially preferred embodimentsabout 95% of the phosphorothioate linkage is in a single stereoisomericform. Most preferably, the phosphorothioate linkage is in a singlestereoisomeric form, substantially free of other stereoisomeric forms.Preferably, the first synthon is in a single stereoisomeric form,substantially free of other stereoisomeric forms.

In some preferred embodiments n is 0. In further preferred embodiments,R¹ groups are subsequently removed to yield new second synthons foriterative synthesis, and chiral auxiliaries are removed after iterativesynthesis is completed. In preferred embodiments of the present methodsthe oligomer of Formula IV contains a plurality of phosphorothioatelinkages.

Preferably, first and second synthons are reacted at a temperature offrom about -20° C. to about 40° C., with from about -15° C. to about 0°C. being more preferred.

In some preferred embodiments the first synthon is formed by reacting acompound of Formula V: ##STR8## with an azaphospholane of Formula VIa:##STR9## wherein R³ --R³ are as defined above; and X is halogen.dialkylamino, imidazole, triazole or substituted phenoxy wherein saidsubstituents are electron withdrawing, preferably halogen or nitro.

In some embodiments the azaphospholane described above is produced byreacting a reagent of formula HO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ and aphosphorus trihalide, phosphorus tri(dialkylamide), phosphorustriphenoxide or phosphorus triimidazolide.

In more preferred embodiments the first synthon is formed by reacting acompound of Formula VII: ##STR10## and a Γ-amino alcohol of formulaHO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷. Preferably, X is chlorine,dialkylamino or diphenoxy, and said reaction is stereoselective. It isespecially preferred that the first synthon is in a singlestereoisomeric form, substantially free of other stereoisomeric forms.

In some preferred embodiments the reaction of first and second synthonsis performed in the presence of a catalyst, said catalyst preferablyhaving one of the Formulas VIII or IX: ##STR11## wherein: R¹² and R¹³are independently hydrogen, halogen, cyano, nitro, alkyl having from oneto 10 carbons, substituted alkyl having from one to 10 carbons, an estergroup, or R¹² and R¹³ together with the carbon atoms to which they areattached, form a substituted or unsubstituted phenyl ring where saidsubstituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to10 carbons, substituted alkyl having from one to 10 carbons, norbornyl,substituted norbornyl, aryl, substituted aryl wherein said substituentsare electron withdrawing, or has the formula: ##STR12## wherein L isprotecting group.

In some preferred embodiments R¹⁴ is halogen or nitro, preferablybromine, and R¹² and R¹³ are each halogen or each cyano, with cyanobeing especially preferred.

Other preferred embodiments R¹⁴ has one of the formulas: ##STR13##wherein R¹⁵ is H, methyl, trialkylsilyl or acetyl.

In some preferred embodiments of the method R³ is cyanomethyl or --CH₂--SO_(k) R⁴ where k is 0, 1 or 2, and R⁷ is lower alkyl or aralkyl.

In further preferred embodiments said first synthon has one of theFormula Xa, XIa, XIIa, XIIIa or XXa: ##STR14## wherein W has theformula: ##STR15## and R¹ --R¹⁶, V, T and Z are as defined above.

Other preferred first synthons have the Formula Xb or Xc: ##STR16##

More preferred first synthons have the Formula XVIIa or XVIIIa:##STR17##

Particularly preferred first synthons have the Formula XIVa: ##STR18##

Especially preferred first synthons have the Formula XVa or XVIa:##STR19##

In preferred embodiments of the methods of the invention R¹ groups areremoved from the oligomers, thus creating new second synthons forfurther iterative synthesis.

Also provided according to the invention are phosphorothioate oligomersproduced by the method of claim 1, and azaphospholanes having FormulaVIb: ##STR20##

In preferred embodiments of the invention, 75% of said azaphospholaneshaving Formula VIb are in a single stereoisomeric form, with 85% beingmore preferred, and 95% being particularly preferred. In especiallypreferred embodiments, the azaphospholanes having Formula VIb are in asingle stereoisomeric form, substantially free of other stereoisomericforms.

In preferred embodiments the azaphospholane has one of the Formulas Xb,XIb, XIIb, Xiiib, Xd, Xe or XXb: ##STR21##

In other preferred embodiments the azaphospholane has the Formula XVIIbor XVIIIb: ##STR22##

In some particularly preferred embodiments the azaphosphalane has theFormula XIVb: ##STR23##

Especially preferred embodiments the azaphospholane has the Formula XVbor XVIb: ##STR24##

Also provided in accordance with the invention are oligomeric compoundscomprising a phosphite linkage having the Formula XXX: ##STR25##

In preferred embodiments of the invention, 75% of said phosphosphitelinkage is in a single stereoisomeric form, with 85% being morepreferred, and 95% being particularly preferred. In especially preferredembodiments, the phosphosphite linkage is in a single stereoisomericform, substantially free of other stereoisomeric forms.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to methods for the synthesis ofphosphorothioate compounds having diastereomerically enrichedphosphorothioate linkages, and to intermediates useful in theirpreparation.

In one aspect, the invention provides methods for the preparation ofphosphorothioate linkages comprising the steps of reacting a firstsynthon of Formula I: ##STR26## wherein: Q is independently O or S;

R¹ is a hydroxyl protecting group;

R² is a chiral auxiliary of formula --C(R⁸)--R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷;

R³ is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl,trihalomethyl, --CH₂ Si(R⁴)₃, or --CH₂ --SO_(k) R⁴ where k is 0, 1 or 2;

R⁴ is independently alkyl, aryl, aralkyl or alkaryl having up to 15carbon atoms;

R⁵ is H, --CN, --Si(R⁴)₃, SO_(k) R⁴ or halogen;

or R⁸ and R¹⁶ are each H, and R³ and R⁵, together, form one of thestructures ##STR27## wherein: R¹⁰ and R¹¹ are H, alkyl having from 1 toabout 10 carbons, --CH₂ C(=O)OR²², --CH₂ CN, --CH₂ Si(CH₃)₃, or o- orp-C₆ H₄ --R²¹ ;

R²¹ is hydrogen, --O--C(=O)CH₃, alkoxy having from 1 to about 10carbons, --NO₂, or --N(R²²)₂ ;

R²² is independently H or alkyl having from one to about 10 carbonatoms;

p is 1 or 2;

Z¹ and Z² are independently halogen, CN, --Si(CH₃)₃, and --C(=O)OR²² ;R³⁰ is hydrogen, --O--C(=O)CH₃, alkoxy having from 1 to about 10carbons, or --O--Si(R₄)₃ ;

R⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

or R⁵ and R⁶, together with the atoms to which they are attached, form a5 or 6 membered ring;

R⁷ is alkyl or aralkyl having up to 15 carbon atoms;

or R⁶ and R⁷, together, form one of the structures ##STR28## wherein V,T, and Z are independently CH or N;

R⁸ is H or methyl;

R¹⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

B is a nucleobase; and

n is an integer from 0 to 50;

with a second synthon of Formula II: ##STR29## wherein R⁹ is a hydroxylprotecting group or a linker connected to a solid support; and

m is an integer from 0 to 50;

for a time and under reaction conditions effective to form a thirdsynthon of Formula III: ##STR30## and contacting said third synthon witha sulfurizing agent to form an oligomer of Formula IV: ##STR31## whereinD is said phosphorothioate linkage having the formula: ##STR32##

In accordance with the invention, first synthons are cyclicphosphoramidites having the general Formula VIc: ##STR33## in which W,R³, R⁵ -R⁸ and R¹⁶ are as defined above.

The reaction of first and second synthons is conducted in the presenceof a catalyst. The structures of the first synthon and the catalyst arechosen such that the opening of the cyclic O--P--N phosphoramidite(azaphospholane) ring proceeds by the steroselective breaking of theintracyclic P--N bond of the azaphospholane, to yield a third synthon,which is diastereomerically enriched at phosphorus. Accordingly, inpreferred embodiments of the methods of the invention, first synthonsare diastereomerically enriched, and more preferably in a singlestereochemical form, substantially free of other stereochemical forms.It is also advantageous for the first synthon and the catalyst to bearsubstituent groups which are of relatively large size (i.e., bulkygroups) to aid in the proper orientation of reactants to achieve thedesired stereoselectivity. As used herein, the term stereoselective hasits normal meaning as a process in which one stereoisomer is produced ordestroyed more rapidly than another, resulting in a predominance of thefavored stereoisomer.

In preferred embodiments catalysts have one of the Formulas VIII or IX:##STR34## wherein R¹² -R¹⁴ are as defined above.

It has been found in accordance with the present invention thatimidazole catalysts having electron-withdrawing substituents, inaddition to substituents of relatively large size, are especiallyadvantageous in production of stereochemically enriched products. Whilenot wishing to be bound by a particular theory, it is believed that thecatalyst first protonates the azaphospholane nitrogen, creating a goodleaving group, which is displaced by the catalyst or its conjugate base.The imidazole or tetrazole attached to the phosphorus is then displacedeither by the 3'-hydroxyl of the nucleosidic species, leading to aphosphite triester of high stereochemical purity, or by the catalyst,leading to epimerization.

It has been found in accordance with the present invention thatcatalysts which have appreciable acidity (i.e., which have pKa values ofabout 2 to 4) and which are relatively large can overcome the tendencytoward epimerization at phosphorus, and result in stereoselectiveaddition of the free 5'-hydroxyl of the nucleosidic species to be added.Thus, preferred substituents for groups R¹³, R¹⁴ and R¹⁵ are those whichare electron withdrawing, (and which therefore increase acidity), and ofa size sufficient to maintain stereoselectivity. It will be recognized,however, that it is not necessary that all three groups R₆, R₇ and R₈ beof great bulk, so long as the overall size of the catalyst is sufficientto afford the desired stereoselectivity. Thus preferred R¹² and R¹³groups are independently hydrogen, halogen, cyano, nitro, alkyl havingfrom one to 10 carbons, substituted alkyl having from one to 10 carbons,an ester group, or R¹² and R¹³, together with the carbon atoms to whichthey are attached, form a substituted or unsubstituted phenyl ring wheresaid substituents are electron withdrawing. Preferred R¹⁴ groups includehydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10carbons, substituted alkyl having from one to 10 carbons, norbornyl,substituted norbornyl, aryl, substituted aryl wherein said substituentsare electron withdrawing, or has the formula ##STR35## wherein L isprotecting group. In preferred embodiments of the invention the catalystis 2,4,5-tribromoimidazole, dibromocyanoimidazole, ordicyanobromoimidazole. In particularly preferred embodiments thecatalyst is 4,5-dicyano-2-bromoimidazole.

It has been found in accordance with the present invetion that thedicyanoimidazole, bromoimidazole, and tribromoimidazole catalystsdescribed in accordance with the present invention are useful assubstitutes for tetrazole catalysts in standard solid phaseoligonucleotide synthetic regimes. Such synthetic procedures are wellknown in the art, and are extensively described in the literature. Seefor example, Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707;4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677and Re. 34,069, and Oligonucleotides and Analogues, A PracticalApproach, Eckstein, F., IRL Press, New York (1991). The use of thecatalysts of the invention in these synthetic methologies providessignificant advantages over tetrazole catalysts, including, for example,significantly lower cost.

In other preferred embodiments R¹⁴ has one of the formulas: ##STR36##wherein R¹⁵ is H, methyl, trimethylsilyl or acetyl.

In some preferred embodiments R⁶ and R⁷, together with the atoms towhich they are attached, form an heterocyclic (i.e., imidazole, triazoleor tetrazole) ring, which performs the function of the catalyst.Preferred first synthons which incorporate the catalyst therein have thegeneral Formula Xa or XIIIa: ##STR37## wherein V, T and Z are eachindependently N or CH. In especially preferred embodiments the firstsynthons incorporate imidazole rings, and have the Formula Xb or Xc:##STR38##

In further preferred embodiments of the invention the imidazole portionsof the first synthons are further substituted, for example, by having aphenyl ring fused thereto. Thus in another preferred embodiment firstsynthons have the Formula XIa: ##STR39##

In further preferred embodiments, first synthons incorporate otherrelatively large substituent groups which facilitate the stereoselectiveopening of the azaphospholane ring. In particularly preferredembodiments first synthons have the Formula XIIa, and particularlyFormula XVIIa or XVIIIa: ##STR40## in which R¹⁰ and R¹¹ are as definedabove.

In especially preferred embodiments, first synthons have the Formula XVbor XVIb: ##STR41##

In some preferred embodiments, the first synthon is obtained by reactionof a compound of Formula V: ##STR42## with an azaphospholane of FormulaVIa: ##STR43## wherein R³ -R⁸ are defined above; and X is halogen,preferably chlorine, dialkylamino, imidazole or substituted phenoxywherein said substituents are electron withdrawing, and preferably arehalogen or nitro.

In more preferred embodiments the first synthon is obtained by reactionof a compound of Formula XII: ##STR44## and a γ-amino alcohol of formulaHO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ; wherein X and R¹ -R¹⁶ are asdefined above.

R₂ is a chiral auxiliary, which has the formula --C(R⁸)R³ --C(R¹⁶)R⁵--CHR⁶ --NHR⁷, and which is formed as a consequence of the opening ofthe cyclic phosphite ring. The chiral auxiliary functions as aprotecting group for the phosphorus linkage during the course of thesynthesis of oligomeric phosphorothioates. Accordingly, chiralauxiliaries are allowed to remain on the growing chain, and are removedat the end of the iterative synthetic regime. Removal of chiralauxiliaries can be conveniently accomplished in a single treatment afterthe completion of the iterative synthesis by treatment with eitheracidic reagents or by base catalyzed β-elimination. Suitable reagentsinclude, for example, 70% trifluoroacetic acid, ammonia, and fluorideion. Removal of chiral auxiliaries via β-elimination should beparticularly advantageous where first synthons have the Formula XXa.

After reacting first and second synthons to form a third synthon, thethird synthon is sulfurized to form a phosphorothioate linkage havingthe formula: ##STR45##

Sulfurization may be accomplished by any of the several sulfurizingagents known in the art to be suitable for conversion of phosphites intophosphorothioates. Useful sulfurizing agents include Beaucage reagentdescribed in e.g., Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L.;3H-1,2-Benzodithiole-3-one 1,1-Dioxide as an Improved SulfurizingReagent in the Solid-Phase Synthesis of OligodeoxyribonucleosidePhosphorothioates, Journal of American Chemical Society, 1990, 112,1253-1254 and Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan J. B.;Beaucage, S. L.; The Automated Synthesis or Sulfur-ContainingOligodeoxyribonucleotides Using 3H-,2-Benzodithiol-3-one 1,1-Dioxide asa Sulfur-Transfer Reagent, Journal of Organic Chemistry, 1990, 55,4693-4699. Tetraethyl-thiuram disulfide can also be used as described inVu, H.; Hirschbein, B. L., Internucleotide Phosphite Sulfurization WithTetraethylthiuram Disulfide, Phosphorothioate Oligonucleotide SynthesisVia Phosphoramidite Chemistry, Tetrahedron Letters, 1991, 32, 3005-3007.Further useful reagents for this step are dibenzoyl Tetrasulfide, Rao,M. V.; Reese, C. B.; Zhengyun, Z., Dibenzoyl Tetrasulphide--A RapidSulphur Transfer Agent in the Synthesis of Phosphorthioate Analogues ofOligonucleotides, Tetrahedron Letters, 1992, 33, 4839-4842;di(phenylacetyl)disulfide, Kamer, R. C. R.; Roelen, H. C. P. F.; van denEist, H.; van der Marel, G. A.; van Boom, J. H., An Efficient ApproachToward the Synthesis of Phosphorothioate Diesters Va the SchonbergReaction, Tetrahedron Letters, 1989, 30, 6757-6760; sulfur; and sulfurin combination with ligands like triaryl, trialkyl or triaralkyl ortrialkaryl phosphines.

The methods of the present invention can also be used to produce analogsof phosphorothioates, including phosphoroselenoates andphosphoroboronates. For example, phosphoroselenoates can be prepared bythe methods of the invention utilizing potassium selenocyanate in placeof the sulfurizing agents described above. Phosphoroboronates can beprepared by similar adaptation of oxidizing agents known known in theart. See, for example, Antisense Research and Applications, Crooke, S.G., and Lebleu, B., Eds. CRC Press, Boca Raton, Fla. (1933).

R₉ and R₁ can each be a hydroxyl protecting group. Protecting groups areknown per se as chemical functional groups that can be selectivelyappended to and removed from functionalities, such as hydroxyl groupsand carboxyl groups. These groups are present in a chemical compound torender such functionality inert to chemical reaction conditions to whichthe compound is exposed. The tert-butyldimethylsilyl (TBDMS) group isrepresentative of protecting groups useful for protecting the hydroxylfunctionality. A preferred protecting group for R¹ is thedimethoxytrityl group. Other representative groups may be found inGreene, T. W. and Wuts, P. G. M., "Protective Groups in OrganicSynthesis" 2d. Ed., Wiley & Sons, 1991. Typically, protecting groups areremoved at the end of the iterative synthesis.

R₉ may alternatively be a linker connected to a solid support. Solidsupports are substrates which are capable of serving as the solid phasein solid phase synthetic methodologies, such as those described inCaruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777;4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re.34,069. Linkers are known in the art as short molecules which serve toconnect a solid support to functional groups (e.g., hydroxyl groups) ofinitial synthon molecules in solid phase synthetic techniques. Suitablelinkers are disclosed in Oligonucleotides And Analogues A PraticalApproach, Ekstein, F. Ed., IRL Press, N.Y, 1991.

Alkyl groups according to the invention include straight chain,branched, and cyclic carbon and hydrogen containing groups such asmethyl, isopropyl, and cyclohexyl groups. Preferred alkyl groups have 1to about 6 carbon atoms.

Aralkyl groups according to the invention include both alkyl and arylportions, although the point of attachment of such groups is through analkyl portion thereof. Benzyl groups provide one example of an aralkylgroup. Alkaryl groups include both alkyl and aryl portions, and areattached through their aryl portions. The term aryl is intended todenote monocyclic and polycyclic aromatic groups including, for example,phenyl, naphthyl, xylyl, pyrrole, and furyl groups. Although aryl groups(e.g., imidazo groups) can include as few as 3 carbon atoms, preferredaryl groups have 6 to about 14 carbon atoms, more preferably 6 to about10 carbon atoms. The alkyl, alkaryl, and aryl groups may be substituted(e.g., i.e, bear halogens and hydroxy groups) or unsubstituted moieties.

Certain substituent groups of compounds of the invention bear electronwithdrawing groups. As used herein, the term "electron wihdrawing" hasits normal meaning as a chemical functionality which electronically orinductively causes the withdrawal of electron density form the moiety towhich the electron withdrawing groups is attached. Representativeelectron withdrawing groups include nitro groups and halogens. Otherelectron withdrawing groups will be apparent to those of skill in theart, once armed with the present disclosure.

Substituent B is a nucleobase. The term nucleobase as used herein isintended to include naturally occurring nucleobases (i.e., heterocyclicbases found in naturally occurring nuclei acids) and their non-naturallyoccurring analogs. Thus, nucleobases according to the invention includenaturally occurring bases adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U), both in their unprotected state and bearingprotecting or masking groups. Examples of nucleobase analogs include N⁴,N⁴ -ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N⁶-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyl uracil, inosine, N⁶ -isopentyladenine,1-methyladenine, 2-methylguanine, 5-methylcytosine, N⁶ -methyladenine,7-methylguanine, 5-methylaminomethyl uracil,5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil, pseudouracil,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil,5-(1-propynyl)-2-thiocytosine, 2-thiocytosine, and 2,6-diaminopurine.Other suitable base analogs, for example the pyrimidine analogs6-azacytosine, 6-azathymidine and 5-trifluoromethyluracil, may be foundin Cook, D. P., et al., International Publication No. 92/02258, which isherein incorporated by reference.

The compounds of the invention are preferably up to 50 nucleobases inlength, with 10 to 30 nucleobases being more preferred, and 15 to 25nucleobases being especially preferred.

In preferred embodiments the phosphorothioate linkage produced by themethod of the invention is diastereomerically enriched. The term"diastereomerically enriched" denotes the predominance of onestereochemical form over the other. In preferred embodiments thephosphorothioate linkage is 75% in a single stereochemical form. Infurther preferred embodiments the phosphorothioate linkage is 85% in asingle stereochemical form, with 90% being further preferred and 95%being especially preferred. In further preferred embodiments thephosphorothioate linkage is in a single stereochemical form,substantially free of other stereochemical forms.

Preferably, following sulfurization, the phosphorothioate is nextconverted to a new first synthon. This is first accomplished by theremoval of the 5'-hydroxyl protecting group R₁, under conditions whichwill necessarily depend upon the chemical identity of the specific R₁group. After removal of the protecting group, the unprotected 5'-alcoholmay be employed as a new second synthon in the iterative method.Libraries of dimeric and higher synthons may be prepared and stored tofacilitate the iterative synthesis of desired nucleobase sequences.

Also provided according to the invention are azaphospholanes of FormulaVIb: ##STR46## where Y is X or W, wherein X is halogen, dialkylamino,imidazole, or substituted phenoxy wherein said substituents are electronwithdrawing, and W has the formula: ##STR47## wherein constituentmembers are as defined above. Preferably, the azaphospholanes of FormulaVIb are diastereomerically enriched. In particular, it is advantageousto have defined stereochemistry around phosphorus atom, to afforddiastereomerically enriched products upon stereoselective opening of theazaphospholane ring.

In preferred embodiments, compounds of the invention have one of theFormulas Xb, XIb, XIIb, XIIIB or XXb: ##STR48## wherein R³ -R¹⁶, Y, V,T, Z, Z₁, Z₂ and p are as defined above.

Particularly preferred embodiments of the compounds of the inventionhave the Formula XIVb, Xd, Xe, XVIIb, XVIIb, XVIIIb, XVb or XVIb:##STR49##

As used herein, the term "contracting" means directly or indirectlycausing placement together of moieties to be contacted, such that themoieties come into physical contact with each other. Contacting thusincludes physical acts such as placing the moieties together in acontainer. The term "reacting" as used herein means directly orindirectly causing placement together or moieties to be reacted, suchthat the moieties chemically combine or transform.

The method of the invention is performed in the presence of a solvent,for example chloroform or acetonitrile. Other solvents suitable for usein the present method will be readily apparent to those skilled in theart, once having been made aware of the present disclosure.

In general, it is preferred that the molar ratio of the catalyst to thefirst synthon starting material be from about 1 to about 50; preferablyfrom about 2.5 to about 10.

The method of the present invention can be carried out in any suitablevessel which provides efficient contacting between the first and secondsynthons, and the catalyst. The reaction vessel used should be resistantto the components of the reaction mixture. Glass-lined vessels would besuitable for this purpose. Additional vessel materials will be apparentto those skilled in the art.

The reagents of the present method may be added in any order. The methodis preferably carried out under an inert atmosphere, any should becarried out in a dry atmosphere. Any suitable inert gas may be employed,such as nitrogen, helium and argon.

Preferably, the method is carried out at temperatures ranging betweenabout -20° C. and about 40° C., with temperatures ranging from about-15° C. to about 0° C. being more preferred.

Reaction time is generally from about one minute to about two hours,with reaction times of from about one minute to about 10 minutes beingpreferred.

Product can be recovered by any of several methods known to those ofskill in the art. Preferably, products are recovered by chromatography.Additional separation of isomers can be accomplished by techniques knownin the art including high performance liquid chromatography.

When R⁹ is a solid support, purification is carried out after removal ofthe oligonucleotide from the solid support using methods known in theart.

The invention is further illustrated by way of the following examples.These examples are illustrative only and are not intended to limit thescope of the appended claims.

EXAMPLES

General methods.

Melting points (m.p.) were determined using an Electrothermal MPapparatus and are uncorrected. Optical rotation measurements werecarried out in the indicated solvents employing a Jasco DIP-140 digitalpolarimeter. Mass spectra (CI or EI) were obtained on an HP 5980Aquadrupole mass spectrometer in the direct-inlet mode.

NMR spectra were recorded on Varian XL200, XL300, or Unity 500spectrometers. Chemical shifts are given in the δ scale in parts permillion. The assignments of proton spectra are based on COSYexperiments. The residual proton signals of deuteriochloroform (δ7.24ppm), methanol (δ3.30 ppm) and acetonitrile (δ1.93 ppm) were used asreference in these solvents. The multiplicities are recorded using thefollowing abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;m, multiplet. ³¹ P NMR spectra were obtained on either a Varian XL200,XL300 or Unity 500 instrument, and chemical shifts are given withrespect of aqueous phosphoric acid. Peak assignments of ¹³ C-NMR spectrawere, in some cases, made with the aid of APT, HMQC or HETCORexperiments.

Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl.Dichloromethane was distilled from P₂ O₅. Triethylamine and acetonitrilewere distilled from CaH₂. N,N-Dimethyl formamide was dried by shakingwith KOH, followed by distillation. Thin-layer chromatography (TLC) wasperformed using Kieselgel 60 F₂₅₄ aluminum-backed plates (0.2 mmthickness) and visualized by UV and/or dipping in a solution of ammoniummolybdate (2.5 g) and ceric sulfate (1 g) in 10% v/v aqueous sulphuricacid (100 ml), followed by heating. Kieselgel 60 (Merck 230-400 mesh)silica gel was employed for column chromatography.

EXAMPLE 1 3R-hydorxy-N-iso-propylbutanoamide (10) ##STR50##

A 2 M solution of trimethylaluminium in hexane (50 ml, 100 mmol) wasslowly added to a solution of 8.5 ml (100 mmol) isopropylamine in 100 mldichloromethane under nitrogen at room temperature. The mixture wasstirred for 30 min and then cooled to 0° C. before 6.6 g (50 mmol) ofethyl-3R-hydroxybutanoate was added. The reaction mixture was stirred atroom temperature for 2 hours for completion, carefully quenched withdilute HCl and extracted with chloroform. The organic extract was driedover MgSO₄ and concentrated to affored 7 g N-isopropyl 3R-hydroxybutanoamide. After recrystallization, 5.5 g pure amide 10 was obtained(yield 76%).: m.p. 62° C.; ¹ H NMR (200 MHz, CDCl₃) δ6.41 (m, 1H, NH),4.35 (b, 1H, OH), 3.78-4.15 (m, 2H, MeCH, Me₂ CH), 2.08-2.32 (m, 2H,CH₂), 1.12 (d, J=6.3 Hz, 3H, Me), 1.07 (d, J=6.6 Hz, 6H, NCHMe₂); ¹³ CNMR (50 MHz, CDCl₃) δ171.5 (C═O), 64.7 (CHOH), 43.8 (NHCH), 41.0 (CH₂),22.7 (Me), 22.4 (NCHMe₂); MS(EI) m/e 145 ([M⁺ ], 25%), 130 (27), 112(4), 101 (6), 86 (34), 69 (8), 58 (22), 44 (100); HRMS(EI) m/e calc'dfor C₇ H₁₅ O₂ N [M⁺ ]: 145.1103, found 145.1109.

EXAMPLE 2R-Hydroxy-4-(N-isopropyl)aminobutane (11) ##STR51##

To a solution of 32 ml 1 M borane (32 mmol) in THF was added 2.32 g (16mmol) 3R-hydroxy-N-iso-propyl-butyl butanoamide 10 in 20 ml THF at 0° C.under nitrogen. The solution was then brought to reflux and maintainedthere for one hours. The reaction mixture was cooled to room temperatureand 1 N HCl was added slowly to quench the reaction. THF was removed invacuo, and the aqueous solution was saturated with solid NaOH and thenwas extracted three times with a total 300 ml chloroform. The combinedchloroform phase was dried, filtered and distilled to afford 1.4 g3-(N-isopropylamino) butan-2-ol 11 as a clear, colorless liquid (yield67%.: ¹ H NMR (200 MHz, CDCl₃) δ3.80-3.96 (m, 1H, CHOH), 2.86-2.98 (m,1H, MeCH), 2.56-2.75 (m, 2H, NCH₂), 1.24-1.60 (m, 2H, CH₂), 1.08 (d,J=6.1 Hz, 3H, Me), 0.98 (d, J=6.2 Hz, 6H, Me₂); ¹³ C NMR (200 MHz,CDCl₃) δ69.5 (OCH), 48.6 (NHCH₂), 46.0 (NHCH), 37.2 (CH₂), 23.5, 22.9,22.5; MS(EI) m/e 131 ([M⁺ ], 10%), 116 (81), 98 (35), 72 (100), 58 (30),56 (45), 44 (37); HRMS(EI) m/e calc'd for C₇ H₁₇ ON [M⁺ ]: 131.1310,found 131.1311; [a]_(D) ²⁰ =32.5° (c=0.21, chloroform).

EXAMPLE 3 2R-Hydroxy-4-(N-tert-butyl)aminobutane (31) ##STR52##

To a solution of 26.4 ml 1 M (26.4 mmol) borane in THF was added 2.1 g(13.2 mmol) 3R-hydroxy-N-tert-butylbutanoamide in 20 ml THF at 0° C.under nitrogen. The solution was then brought to reflux and maintainedthere for one hours. The reaction mixture was cooled down to roomtemperature and 1 N HCl was added slowly to quench the reaction. THF wasremoved in vacuo, and the aqueous solution was saturated with solid NaOHand then was extracted three times with a total 250 ml diethyl ether.The combined organic phase was dried, filtered and distilled to afford398 mg 2R-hydroxy-4-(N-tert-butyl)aminobutane 31 as a clear, colorlessliquid (yield 20.1%): ¹ H NMR (200 MHz, CDCl₃) δ3.80-3.92 (m, 1H, CHOH),3.40-3.80 (b, 2H, OH, NH), 2.50-2.90 (m, 2H, NCH₂), 1.28-1.61 (m, 2H,CH₂), 1.07 (d, 3H, Me), 1.03 (s, 9H, Me₃)

EXAMPLE 4 5'--O--(tert-butyldimethylsily) thymidine (1) ##STR53##

To a solution of 2.42 g (10 mmol) thymidine in 15 ml DMF was added 1.7 g(25 mmol) imidazole and 1.6 g (10.6 mmol) tert-butyldimethyl silylchloride. The solution was stirred at room temperature for 3 hours. DMFwas then removed in vacuo and the residue was dissolved in 150 ml ofethyl acetate. The solution was washed with water and the organic layerwas dried over MgSO₄. After removing the solvent, the solid wasrecrystallized with ethyl acetate/pentane to obtain 2.5 g pure5'--O--(tert-butyldimethylsilyl) thymidine 1 (70% yield).: m.p. 193-194°C.; ¹ H NMR (500 MHz, CDCl₃) δ9.0 (s, 1H, NH), 7.50 (s, 1H, H-6), 6.36(dd, J=5.8, 8.1 Hz, 1H, H-1'), 4.44 (m, 1, H-3'), 4.03 (m, 1H, H-4'),3.85 (m, 2H, H-5'), 2.66 (d, J=3.8 Hz, 1H, OH), 2.35 (m, 1H, H-2'), 2.07(m, 1H, H-2'), 1.89 (s, 3H, C═CMe), 0.89 (s, 9H, CMe₃), 0.09 (s, 6H,SiMe₂); ¹³ C NMR (125 MHz, CDCl₃) δ163.8 (C-4), 150.4 (C-2), 135.4(C-6), 110.9 (C-5), 87.2 (C-4'), 85.0 (C-1'), 72.6 (C-3'), 63.6 (C-5'),41.1 (C-2'), 25.9 (SiCMe₃), 18.3 (SiCMe₃), 12.5 (C═CMe), -5.4 (SiMe₂),-5.5 (SiMe₂).

EXAMPLE 5 5'--O--(4,4'-dimethyoxytrityl) thymidine (16) ##STR54##

Triethylamine (10 ml) in 200 ml THF was injected into a solid mixture of6.8 g (28.0 mmol) thymidine and 10.2 g (28.6 mmol) 4,4'-dimethyoxytritylchloride under nitrogen with stirring. The solution was stirred at roomtemperature for 2 hours. After completion of the reaction, 10 mlmethanol was added to consume the excess DMTrCl. The mixture was stirredfor 5 minutes and the solvent removed by rotary evaporation. The residuewas dissolved in 250 ml of ethyl acetate and the solution was washedwith saturated NaHCO₃ and dried over MgSO₄. The solid was recrystallizedfrom ethyl acetate/hexane to obtain 13.0 g 5'-protected thymidine 16(85.6%).: m.p 124-126° C.; ¹ H NMR (200 MHz, CDCl₃) δ8.97 (s, 1H, NH),7.60 (m, 1H, H-6), 6.72-7.42 (m, 13H, Ph), 6.42 (m, 1H, H-1'), 4.56 (m,1H, H-3'), 4.05 (m, 1H, H-4'), 3.78 (s, 6H, OMe₂), 3.41 (m, 2H, H-5'),2.62 (m, 1H, OH), 2.46 (m, 2H, H-2'), 1.46 (s, 3H, Me); ¹³ C NMR (50MHz, CDCl₃) δ163.0 (C-4), 157.6, 149.8 (C-2), 143.5, 135.0 (C-6), 134.7,134.6, 129.4, 127.4, 127.3, 126.5, 112.8, 110.9 (C-5), 86.7 (C-4'),86.2, 84.7 (C-1'), 72.5 (C-3'), 63.8 (C-5'), 55.5 (OCH₃), 41.3 (C-2'),12.5 (CH₃).

EXAMPLE 6 5'--O--(4,4'-dimethoxytrityl)-3'--O--(tert-butyldimethylsilyl)thymidine (17) ##STR55##

To a solution of 13.0 g (23.9 mmol)5'--O--(4,4'-dimethoxytrityl)thymidine 16 in 50 ml DMF was added 3.0 g(44 mmol) imidazole and 3.6 g (23.9 mmol) tert-butyl-dimethylsilylchloride. The solution was stirred at room temperature for 3 hours. DMFwas then removed in vacuo and the residue was dissolved in 300 ml ofethyl acetate. The solution was washed with water and the organic layerwas dried over MgSO₄. After concentration of the solution andrecrystallization from ethyl acetate/hexane, the solid product5'--O--(4,4'-dimethoxy)-3'-(tert-butyldimethylsilyl)thymidine 17 wasused directly for the next reaction.

EXAMPLE 7 3'--O--(tert-butyldimethylsilyl)thymidine (18) ##STR56##

A solution of 5 g (7.6 mmol)5'--O--(4,4'-dimethoxytrityl)-3'--O--(tert-butyldimethylsilyl) thymidine17 in 100 ml 80% aq. acetic acid was stirred until the removal ofdimethyoxytrityl group was completed. Saturated Na₂ CO₃ was then addedto adjust the pH of the solution to 6-7. The solution was then extractedwith ethyl acetate. The extract was dried and the mixture waschromatographed on a silica gel column (CH₂ Cl₂ :MeOH=20:1) to give 2.5g of 3'--O--(tert-butyldimethylsilyl) thymidine 18 (92.6%).: m.p. 93-95°C. (lit. 83-84° C.); H NMR (200 MHz, CDCl₃) δ9.18 (s, 1H, NH), 7.36 (b,1H, H-6), 6.12 (t, J=6.8 Hz, 1H, H-1'), 4.44-4.48 (m, 1H, H-3'),3.69-3.91 (m, 3H, H-4', H-5'), 2.87 (m, 1H, OH), 2.15-2.35 (m, 2H,H-2'), 1.87 (s, 3H, C═CMe), 0.86 (s, 9H, CMe₃), 0.05 (s, 6H, SiMe₂); ¹³C NMR (125 MHz, CDCl₃) δ163.9 (C-4), 150.4 (C-2), 137.1 (C-6), 110.9(C-5), 87.6 (C-4'), 86.8 (C-1'), 71.5 (C-3'), 61.9 (C-5'), 40.4 (C-2'),25.7 (SiCMe₃), 17.9 (SiCMe), 12.5 (C═CMe), -4.7 (SiMe₂), -4.9 (SiMe₂).

EXAMPLE 82-Chloro-3-iso-propyl-6R-methyl-1-oxa-3-aza-2-phosphacyclohexane (12)##STR57##

To a solution of 2.2 ml (3.45 g, 25 mmol) phosphorus trichloride in 30ml dichloromethane was added a solution of 2.89 g (22 mmol)2R-hydroxy-4-(N-iso-propyl)aminobutane 11 and 5.0 g (6.9 ml, 50 mmol)triethylamine in 20 ml dichloromethane with vigorous stirring undernitrogen at 0° C. Stirring was continued at room temperature for 0.5hour. The solvent was removed by evaporation under reduced pressure andthe residue was extracted with diethyl either (3×50 ml). Distillationgave 2.9 g of product 12 (74.4% yield).: ¹ H NMR (200 MHz, CDCl₃)δ4.48-4.68 (m, 1H, OCH), 3.40-3.61 (m, 1H, NCH), 3.15-3.38 (m, 1H,NCH₂), 2.80-2.96 (m, 1H, NCH₂), 1.71-1.85 (m, 2H, CH₂), 1.25 (d, J=6.4Hz, 3H, Me), 1.13 (dd, 6H, Me₂); ¹³ C NMR (50 MHz, CDCl₃) δ69.5 (d,J=4.3 Hz, OCMe), 49.6 (d, J=34 Hz, NCMe₂), 37.4 (d, J=5.5 Hz, NCH₂),33.4 (CH₂), 22.2 (d, J=4.3 Hz, Me), 21.1 (d, J=13.3 Hz, Me₂), 19.2 (d,J=5.0 Hz, Me₂); ³¹ P NMR (81 MHz, CDCl₃) δ160.6.

EXAMPLE 9 Phosphoramidite (13a) ##STR58##

To a solution of 215 mg (1.1 mmol) of2-chloro-3-isopropyl-6R-methyl-1-oxa-3-aza-2-phosphacyclohexane 12 in 40ml CH₂ Cl₂ was added a solution of 356 mg (1.0 mmol) of5-O-(tert-butyldimethylsilyl)thymidine and 0.154 ml triethylamine (111mg, 1.1 mmol) in 20 ml CH₂ Cl₂ with vigorous stirring under nitrogen at0° C. The mixture was stirred at room temperature until TLC indicatedthat the reaction had gone to completion. The reaction mixture wastransferred to 150 ml ethyl acetate, which was previously washed with asaturated NaHCO₃ solution. Saturated NaHCO₃ was then added to wash thesolution. The separated organic phase was dried over MgSO₄. A white foam(quantitative yield) was formed after the solvent was removed byevaporation under reduced pressure. Two components were found from ³¹ PNMR spectra in a ratio of 1:3 (135.0 ppm:133.6 ppm). The reactionmixture in CDCl₃ was then refluxed for about 4 hours to get a ratio ofup to 1:12 (135.0 ppm:133.6 ppm). After chromatography on a silica gelcolumn (hexane:ethyl acetate:triethylamine=5:3:2), the fast elutingcomponent (133.6 ppm) was separated as a pure diastereomer.: ¹ H NMR(500 MHz, CDCl₃) δ7.50 (m, 1 H, H-6), 6.36 (dd, J=8.5, 5.8 Hz, 1 H,H-1'), 4.56 (m, 1 H, H-3'), 4.30 (m, 1 H, HCMe), 4.04 (m, 1 H, H-4'),3.75-3.90 (m, 2 H, H-5'), 3.39 (hept, J=6.3 Hz, 1 H, NCH), 3.24 (m, 1 H,NCH₂), 2.72 (m, 1 H, NCH₂), 2.35 (m, 1 H, H-2'), 2.06 (m, 1 H, H-2'),1.89 (d, J=0.9 Hz, 3 H, CH₃), 1.64 (m, 2 H, CH₂), 1.16 (d, J=6.4 Hz, 3H, OCHMe), 1.08 (dd, J=2.0 Hz, 6.3 Hz, 6 H, NCMe₂), 0.89 (s, 9 H,SiCMe₃), 0.09 (d, J=1.5 Hz, 6 H, SiMe₂); ¹³ C NMR (125 MHz, CDCl₃)δ163.9 (C-4), 150.5 (C-2), 135.3 (C-6), 110.9 (C-5), 86.5 (d, J=2.8 Hz,C-4'), 84.8 (C-1'), 73.3 (d, J=19.2 Hz, C-3'), 66.0 (d, J=2.7 Hz, OCH),63.2 (C-5'), 49.2 (d, J=34.8 Hz, NCH), 40.4 (d, J=4.6 Hz, C-2'), 36.6(d, J=5.5 Hz, NCH₂), 34.6 (CH₂), 25.9 (SiCMe₃), 22.9 (d, J=4.6 Hz,OCMe), 21.8 (d, J=10 Hz, NCMe₂), 21.0 (d, J=4.6 Hz, NCMe₂), 18.3(SiCMe₃), 12.5 (C═CMe), -5.5 (SiMe₂), -5.4 (SiMe₂); ³¹ P NMR (81 MHz,CDCl₃) δ133.6; MS (CI, NH₃)) m/e 516 ([M+H⁺ ], 37%), 390 (21), 339 (92),178 (100), 160 (55); HRMS (CI, NH₃) m/e calc'd for C₂₃ H₄₃ N₃ O₆ PSi[M+H⁺ ]: 516.2659, found 516.2664.

EXAMPLE 10 Phosphoramidite (13b) ##STR59##

To a solution of 215 mg (1.1 mmol)2-chloro-3-isopropyl-6R-methyl-1-oxa-3-aza-2-phosphocyclohexane 12 in 40ml CH₂ Cl₂ was slowly added a solution of 356 mg (1.0 mmol)5'-O-(tert-butyldimethylsilyl)thymidine 1 and 0.154 ml triethylamine(111 mg, 1.1 mmol) in 20 ml CH₂ Cl₂ with vigorous stirring undernitrogen at -78° C. The mixture was stirred at -78° C. until TLCindicated that the reaction had gone to completion. The reaction mixturewas quickly transferred to 150 ml ethyl acetate, which was previouslywashed with a cold saturated NaHCO₃ solution. Cold saturated NaHCO₃ wasthen added to wash the solution. The separated organic phase was driedover MgSO₄. A white foam (quanitative yield) was formed after thesolvent was removed by evaporation under reduced pressure at 0° C. Twocomponents were found from ³¹ P NMR spectra in a ratio of 5:1 (135.1ppm:133.6 ppm). The reaction mixture in CDCl₃ was immediatelychromatographed on a silica gel column(hexane:ethylacetate:triethylamine=5:3:2) and the slow eluting component(135.1 ppm) was separated as an enriched diastereomer (92% pure).: ¹ HNMR (500 MHz, CDCl₃) δ7.48 (m, 1 H, H-6), 6.32-6.35 (dd, J=5.4, 7.8 Hz,1 H, H-1'), 4.55-4.60 (m, 1 H, H-3'), 4.02-4.10 (m, 1 H, OCH), 4.00 (m,1 H, H-4'), 3.74-3.89 (m, 2 H, H-5'), 3.36-3.45 (m, 1 H, NCH), 3.09-3.14(m, 1 H, NCH₂), 2.85-2.93 (m, 1 H, NCH₂), 2.40-2.44 (m, 1 H, H-2'),2.12-2.21 (m, 1 H, CH₂), 2.00-2.06 (m, 1 H, H-2'), 1.88 (s, 3 H,C═CCH₃), 1.81-1.86 (m, 1 H, CH₂), 1.28 (d, J=6.3 Hz, 3 H, OCMe), 1.11(dd, J=6.3, 11.7 Hz, 6 H, NCMe₂), 0.89 (s, 9 H, Me₃), 0.08 (d, J=2.0 Hz,6 H, SiMe₂); ¹³ C NMR (125 MHz, CDCl₃) δ163.7 (C-4), 150.2 (C-2), 135.5(C-6), 110.8 (C-5), 87.0 (d, J=6.4 Hz, C-4'), 84.8 (d, J=5.5 Hz, C-1'),72.8 (d, J=19.2 Hz, OCHMe), 70.3 (d, 7.3, C-3'), 63.1 (C-5'), 49.5 (d,J=39.4 Hz, NCHMe₂), 40.1 (d, J=1.8 Hz, C-2'), 36.9 (d, J=5.5 Hz, NCH₂),31.5 (d, J=7.3 Hz, CH₂), 25.9 (SiCMe₃), 23.3 (OCMe), 21.7 (d, J=11.0 Hz,NCHMe₂), 21.1 (d, J=5.5 Hz, NCHMe₂), 18.4 (SiCMe₃), 12.5 (C═CMe), -5.4(SiMe₂), -5.5 (SiMe₂); ³¹ P NMR (81 MHz, CDCl₃) δ135.1

EXAMPLE 11 Phosphoroamidite (14) ##STR60##

To a solution of 0.2 ml (315 mg, 2.3 mmol) phosphorus trichloride in 10ml dichloromethane was added a solution of 320 mg (2.2 mmol) butanol 31and 0.7 ml triethylamine in 5 ml dichloromethane with vigorous stirringunder nitrogen at 0° C. and then at room temperature for 0.5 hour. Thesolvent was removed by evaporation under reduced pressure and theresidue was extracted with diethyl ether (2×40 ml). After removing theether, 512 mg residual oil was obtained. Then 209 mg (1.0 mmol) of thisresidual oil was dissolved in 10 ml dichloromethane, a solution of 200mg (0.56 mmol) 5'-O-(tert-butyldimethylsilyl)thymidine 1 and 0.16 mltriethylamine (1.1 mmol) in 10 ml CH₂ Cl₂ was added with vigorousstirring under nitrogen at room temperature. The mixture was stirred atroom temperature until TLC indicated that the reaction had gone tocompletion. The reaction mixture was transferred to 100 ml ethylacetate, which was previously washed with saturated NaHCO₃ solution.Saturated NaHCO₃ and then water was added to wash the solution. Theseparated organic phase was dried over MgSO₄. A white foam (quantitativeyield) was formed after the solvent was removed by evaporation underreduced pressure. Two components were found from ³¹ P NMR spectra in aratio of 1:5 (132.4 ppm:130.8 ppm). The reaction mixture in C₆ D₆ wasthen refluxed for about 4 hours to get a ratio of up to 1:9 (d 137.8ppm:136.1 ppm in benzene).

EXAMPLE 12 Phosphoramidite (15) ##STR61##

To a solution of 6.30 g (4.0 ml, 46 mmol) phosphorus trichloride in 100ml dichloromethane was added a solution of 6.61 g (40 mmol) (1R, 2S)ephedrine and 10.1 g (14 ml, 100 mmol) triethylamine in 50 mldichloromethane with vigorous stirring under nitrogen at 0° C. and thenat room temperature for 0.5 hour. The solvent was removed by evaporationunder reduced pressure and the residue was extracted with diethyl ether(3×100 ml). After removing the ether, 9.3 g residual oil was obtained.Then 345 mg (1.5 mmol) of this residual oil was dissolved in 40 mldichloromethane, a solution of 356 mg (1.0 mmol)5'-O-(tert-butyldimethylsilyl)thymidine 1 and 0.154 ml triethylamine(111 mg, 1.1 mmol) in 20 ml CH₂ Cl₂ was added with vigorous stirringunder nitrogen at -78° C. The mixture was stirred at -78° C. until TLCindicated that the reaction had gone to completion. The reaction mixturewas quickly transferred to 150 ml ethyl acetate, which was previouslywashed with cold saturated NaHCO₃ solution. Cold saturated NaHCO₃ wasadded to wash the solution. The separated organic phase was dried overMgSO₄. A white foam (quantitative yield) was formed after the solventwas removed by evaporation under reduced pressure at 0° C. Twocomponents were found from the ³¹ P NMR spectrum in a ratio of 1:2(147.3 ppm:141.6 ppm). The reaction mixture in CDCl₃ was then refluxedfor about 4 hours to get a ratio of up to 1:5 (151.5 ppm:143.5 ppm).After chromatography on silica gel column (hexane:ethylacetate:triethylamine=5:3:2), the slow eluting component (143.5 ppm) wasseparated as a pure diastereomer.: ¹ H NMR (500 MHz, CDCl₃) δ7.23-7.50(m, 5 H, Ph), 6.35 (m, 1 H, H-1'), 5.60 (d, J=6.8 Hz, 1 H, PhCH),4.71-4.75 (m, 1 H, H-5'), 4.01-4.02 (m, 1 H, H-4'), 3.78-3.87 (m, 2 H,H-5'), 3.52-3.58 (m, 1 H, MeCH), 2.64 (d, J=12.2 Hz, 3 H, NMe),2.31-2.36 (m, 1 H, H-2'), 2.05-2.11 (m, 1 H, H-2'), 1.90 (s, 3 H,C═CMe), 0.90 (s, 9 H, CMe₃), 0.60 (d, J=6.4 Hz, 3 H, CHMe), 0.09 (s, 6H, SiMe₂); ¹³ C NMR (125 MHz, CDCl₃) δ163.9 (C-4), 150.5 (C-2), 137.9(Ph), 135.3 (C-6), 128.1 (Ph), 127.7 (Ph), 126.7 (Ph), 111.0 (C-5), 86.7(d, J=1.8 Hz, C-4'), 84.7 (OCPh), 84.6 (d, J=9.2 Hz, C-1'), 73.1 (d,J=18.3 Hz, C-3'), 63.0 (C-5'), 57.5 (d, J=5.6 Hz, NCMe), 40.6 (d, J=5.5Hz, C-2'), 28.8 (d, J=17.4, NCH₃), 25.9 (SiCMe₃), 18.3 (SiCMe₃), 14.6(d, J=3.7 Hz, NCHMe), 12.5 (C═CMe), -5.4 (SiMe₂), -5.5 (SiMe₂); ³¹ P NMR(81 MHz, CD₃ CN) δ143.5

EXAMPLE 13 Phosphite triester (20a) from phosphoramidite 13a with MeOH##STR62##

Diastereomeric pure phosphoramidite 13a (10 mg, fast eluting component)in 0.5 ml CDCl₃ was put in 5 mm NMR tube, and 50 μl of dry MeOH (a largeexcesses) was then added by syringe, followed by 1 mg ofdicyanoimidzole. The reaction was monitored by ³¹ P NMR until thereaction went to completion. The phosphite triesters formed in a ratioof 10:1 (139.4 ppm:138.8 ppm) and were purified on a silica gelcolumn.:¹ H NMR (500 MHz, CDCl₃) δ7.48 (s, 1 H, H-6), 6.35 (dd, J=5.4,8.8 Hz, 1 H, H-1'), 4.78-4.82 (m, 1 H, H-3'), 4.24-4.33 (m, 1 H, OCH),4.10 (d, J=2.4 Hz, 1 H, H-4'), 3.76-3.89 (m, 2 H, H-5'), 3.50 (d, J=10.7Hz, 3 H, CH₃), 2.78 (m, 1 H, NCH), 2.65 (m, 2 H, NCH₂), 2.40 (m, 1 H,H-2'), 2.04 (m, 1 H, H-2'), 1.89 (d, J=1.0, Hz, 3 H, C═C--CH₃),1.64-1.78 (m, 2 H, OCHCH₂), 1.25 (d, J=6.4 Hz, 3 H, OCHCH₃), 1.03 (d,J=6.3 Hz, 6 H, NCHMe₂), 0.91 (s, 9 H, SiCMe₃), 0.10 (d, J=2.0 Hz, 6 H,SiMe₂); ¹³ C NMR (125 MHz, CDCl₃) δ163.5 (C-4), 150.1 (C-2), 135.3(C-6), 110.9 (C-5), 86.5 (d, J=2.7 Hz, C4'), 84.8 (C-1'), 72.1 (d, J=8.2Hz, C-3'), 69.5 (OCH), 63.0 (C-5'), 49.2 (d, J=9.2 Hz, OCH₃), 48.8 (Me₂CNH), 40.3 (d, J=3.7 Hz, C-2'), 38.7 (d, J=4.6 Hz, CH₂), 25.9 (SiCMe₂),22.9 (d, J=3.7 Hz, NCHMe₂), 18.3 (SiCMe₂), 12.5 (C═CMe), -5.4 (SiMe₂);³¹ P NMR (121 MHz, CDCl₃) δ139.4

EXAMPLE 14 Phosphite triester (20b) from Phosphoramidite 13b withMethanol ##STR63##

Diastereomeric enriched (75%) phosphoramidite 13b (10 mg, slow elutingcomponent) in 0.5 ml CDCl₃ was put in 5 mm NMR tube, and 50 μl dry MeOH(a large excess) was then added by syringe, followed by 1 mg ofdichloroimidazole. The reaction was monitored by ³¹ P NMR until thereaction went to completion. The phosphite triesters formed in a ratioof 1:2 (139.4 ppm:138.8 ppm) were purified on a silica gel column. ¹ HNMR (500 MHz, CDCl₃) δ7.48 (s, 1 H, H-6), 6.34 (m, 1 H, H-1'), 4.78-4.82(m, 1 H, H-3'), 4.25-4.33 (m, 1 H, OCH), 4.11 (d, J=2.4 Hz, 1 H, H-4'),3.77-3.89 (m, 2 H, H-5'), 3.49 (d, J=10.3 Hz, 3 H, CH₃), 2.76 (m, 1 H,NCH), 2.64 (m, 2 H, NCH₂), 2.39 (m, 1 H, H-2'), 2.05 (m, 1 H, H-2'),1.90 (s, 3 H, C═C--CH₃), 1.65-1.78 (m, 2 H, OCCH₂), 1.26 (d, J=6.4 Hz, 3H, OCHCH₃), 1.04 (d, J=6.4 Hz, 6 H, NCHMe₂), 0.91 (s, 9 H, SiCMe₃), 0.10(d, J=2.0 Hz, 6 H, SiMe₂); ³¹ P NMR (121 MHz, CDCl₃) δ138.8

EXAMPLE 15 Phosphite triester (32) from Phosphoramidite 13a withn-Butanol ##STR64##

Diastereomerically pure phorphoramidite 13a (10 mg, fast elutingcomponent) in 0.5 ml CDCl₃ was placed in 5 mm NMR tube, and 50 μl dryn-butanol (a large excess) was then added by syringe, followed by 1 mgof dicyanoimidazole. The reaction was monitored by ³¹ P NMR until thereaction went to completion. The phosphite triesters formed in a ratioof 7:1 (138.9 ppm:138.5 ppm) were purified on a silica gel column. ¹ HNMR (500 MHz, CDCl₃), δ7.49 (d, J=1.5 Hz, 1 H, H-6), 6.35 (dd, J=5.5,8.8 Hz, 1 H, H-1'), 4.83 (m, 1 H, H-3'), 4.24-4.31 (m, 1 H, OCH), 4.11(d, J=2.0 Hz, 1 H, H-4'), 3.71-3.89 (m, 4 H, 2 H-5', OCH₂), 2.78 (m, 1H, NCH), 2.66 (m, 2 H, NCH₂), 2.39 (m, 1 H, H-2'), 2.02 (m, 1 H, H-2'),1.89 (s, 3 H, C═C--CH₃), 1.63-1.77 (m, 2 H, OCMeCH₂), 1.57 (m, 2 H,CH₂), 1.36 (m, 2 H, CH₂), 1.24 (d, J=6.3 Hz, 3 H, OCHCE₃), 1.04 (d,J=6.3 Hz, 6 H, NCHMe₂), 0.91 (s, 9 H, SiCMe₃), 0.10 (d, J=2.0 Hz, 6 H,SiMe₂); ³¹ P NMR (121 MHz, CDCl₃) δ138.9.

EXAMPLE 16 Phosphite triester (33a) from phosphoramidite 7a withMethanol ##STR65##

Diastereomerically pure phosphoramidite 7a (10 mg, fast elutingcomponent) in 0.5 ml CDCl₃ was put in 5 mm NMR tube, and 50 μl drymethanol (a large excess) was then added by syringe, followed by 1 mg of4,5-dicyano-2-bromoimidazole. The reaction was monitored by ³¹ P NMRuntil the reaction went to completion. The phosphite triester consistedof only one diastereomer (139.1 ppm) and was purified on a silica gelcolumn. ¹ H NMR (500 MHz, CDCl₃) δ7.48 (s, 1 H, H-6), 6.34 (dd, J=5.4,8.8 Hz, 1 H, H-1'), 4.78 (m, 1 H, H-3'), 4.09 (m, 1 H, H-4'), 3.76-3.89(m, 4 H, 2 H-5', OCH₂), 3.50 (d, J=10.2 Hz, 3 H, OCH₃), 2.80 (m, 1 H,NCHMe₂), 2.67 (t, J=6.8 Hz, 2 H, NHCH₂), 2.39 (m, 1 H, H-2'), 2.05 (m, 1H, H-2'), 1.90 (s, 3 H, C═C--CH₃), 1.78 (m, 2 H, OCHCH₂), 1.03 (d, J=6.3Hz, 6 H, NCHMe₂), 0.91 (s, 9 H, SiCMe₃), 0.10 (s, 6 H, SiMe₂); ³¹ P NMR(121 MHz, CDCl₃) δ139.1; MS(CI) m/e: 534 ([M+H⁺ ], 100%), 502 (14.4),376 (13.6), 339 (74.6), 281 (20.4), 164 (59.8)

EXAMPLE 17

Phophite triester (33b) from phosphoramidite 7b with Methanol ##STR66##

Diastereomerically enriched (92%) phosphoramidite 7b (10 mg, sloweluting component) in 0.5 ml CDCl₃ was placed in 5 mm NMR tube, and 50μl dry methanol (a large excess) was then added by syringe, followed by1 mg of dicyanobromoimidazole. The reaction was monitored by ³¹ P NMRuntil the reaction went to completion. Diastereomerically enrichedphosphite triester 33b (92%) in ratio of 1:11 (139.2 ppm:138.8 ppm) waspurified on a silica gel column. ⁻ P NMR (121 MHz, CDCl₃) δ138.8

EXAMPLE 18

Thiophosphonate (44) ##STR67##

Diastereomerically pure 13a (100 mg, fast eluting component) wasdissolved in 5 ml CDCl₃, and 0.5 ml MeOH was then added by syringe,followed by 5 mg of 2-bromo-4,5-dicyanoimidazole. The reaction wasmonitored by ³¹ P NMR until the reaction went to completion with almostonly one diastereomer of phosphite triester 20a (139.4 ppm) being found,and then 10 mg sulfur was added. Within 5 minutes, the sulfurizationwent to completion. After chromatography (ethylacetate:triethylamine=1:1), sulfurized product 24 was obtained in anoily form. ¹ H NMR (500 MHz, CDCl₃) δ7.50 (d, J=1.4 Hz, 1H, H-6). 6.36(dd, J=5.4, 9.3 Hz, 1H, H-1'), 5.08 (m, 1H, H-3'), 4.63-4.72 (m, 1H,OCH), 4.25 (m, 1H, H-4'), 3.88 (m, 2H, H-5'), 3.37 (d, J=13.7 Hz, 3H,OCH₃), 2.77 (m, 1H, NCHMe₂), 2.67 (t, J=6.4 Hz, 2H, NCH₂), 2.48 (m, 1H,H-2'), 2.09 (m, 1H, H-2'), 1.90 (d, J=1.5 Hz, 3H, C═C--CH₃), 1.70-1.86(m, 2H, OCCH₂), 1.32 (d, J=5.9 Hz, 3H, OCHCH₃), 1.04 (dd, J=2.9, 5.9 Hz,6H, NCHMe₂), 0.92 (s, 9H, SiCMe₃), 0.12 (s, 6H, SiMe₂); ³¹ P NMR (121MHz, CDCl₃) δ67.5

EXAMPLE 19

2-Bromo-4,5-dicyanoimidazole (21) ##STR68##

To 1.18 g (10 mmol) 4,5-dicyanoimidazole and 25 ml 0.1 M NaOH was added1.8 ml Br₂ (35 mmol). The mixture was stirred overnight at roomtemperature and then acidified with dilute HCl. The solid was filtered,rinsed with water and recrystallized from water to give 1.5 g ofdicyanobromoimidazole 21 (yield 76.4%).: m.p. 147-149° C. (lit. 141-143°C.); Rf=0.65 (ethyl acetate:methanol=4:1); MS (EI): 198 ([M+2], 96%),196 ([M⁺ ], 100%, 171 (28.5), 169 (29.2), 117 (27.4), 91 (19.0), 64(20.6), 53 (22.4), 38 (18.8).

EXAMPLE 20

Synthesis of (S)-methyl-3-(5-imidazolyl)-2-hydroxypropionate (3)hydrochloride ##STR69##

(L)-Histidine (3.103 g, 20 mmol) was first dissolved in 30 ml of 1 Nhydrochloric acid solution. This solution was cooled down to 0° C., thena solution of sodium nitrite (2.070 g, 30 mmol) in 10 ml distilled waterwas added dropwise over a period of 1 hour. The solution was stirredovernight at 0° C., then evaporated to dryness in vacuo with heating. 20ml of distilled water was added to the solid residue, the mixture wasevaporated once more with toluene in order to azeotropically remove thewater residue as much as possible. After drying the compound in highvacuum overnight and without isolation of the intermediate acid, themixture was dissolved into 50 ml of dry methanol and stirred under Ar.This solution was cooled down to 0° C. and a stream of gaseous hydrogenchloride was bubbled through the mixture. After 1.5 hours, TLC indicateddisappearance of the acid and the reaction was stopped. The mixture wasevaporated in vacuo with heating to yield a sticky yellow solid thatcould be crystallized from a mixture of ethanol and ether to yield 3.10g of (2).HCl, m.p. 139-142° C., [α]_(s) -21° (c 1.9, methanol, 25° C.)(litt. -22°) ¹ HNMR (200 MHz, CD₃ OD) δ8.90 (s, 1H, NCHN); 7.30 (s, 1H,NCHC); 4.40 (dd, ABX, ¹ J_(Ha-Hx) =5.40 Hz, ³ J_(Hb-Hx) =5.00 Hz, CHOH);3.72 (s, 3H, OCH₃); 2.85-3.10 (ABX, 2H, ², J_(Ha-Hb) =13.75 Hz, ³J_(Hb-Hx) =5.0 Hz, ³ J_(Ha-Hx) =5.4 Hz, CH₂); ¹³ C NMR (non decoupled)(125 MHz, D₂ O) δ175.3 (s,CO); 134.2 (d, NCHN); 129.7 (s, CH₂ CN); 117.6(d, CCHN); 69.9 (d, CHOH); 53.6 (q, CH₃); 29.5 (t,CH₂); M.S. (M+1)⁺ 171

EXAMPLE 21

Synthesis of imidazo-oxazaphospholidine (4) ##STR70##

In scrupulously dry glassware, compound (3) (0.236 g, 1.39 mmol) wadadded and dried in vacuo overnight, then put under an atmosphere of Ar.This compound was suspended into 5 ml of dry ether, then triethylamine(0.20 ml, 3.15 mmol) was added. The suspension was cooled down to 0° C.and stirred under Ar. Then, methyl dichlorophosphite (0.15 ml, 1.58mmol) was syringed into the mixture quickly. As soon as the phosphitewas introduced, a thick white precipitate was observed, corresponding tothe formation of triethylammonium chloride. After 15 min, ³¹ P NMRshowed several signals between 0.176 and 120 ppm, as well as after 2 to4 hours. After overnight stirring at room temperature, ³¹ P NMR showed asingle signal at 143.5 ppm. Compound decomposed upon trying to handle it(dilution in dry ether, filtration in an Ar atmosphere, concentration byevaporation of the ether) as indicated by ³¹ P NMR by several peaks at5-20 ppm, corresponding most likely to H-phosphonates derivatives.Changing the reaction conditions did not bring any improvement.

Therefore, compound (4) could not be further purified and analyzed.

EXAMPLE 22

Synthesis of 1-tritylimidazole (5) ##STR71##

To a solution of trityl chloride (5.58 g, 20.0 mmol.) in dry methylenechloride (100 ml) cooled down to 0° C. and stirred under Ar, was addeddropwise over 1.5 hours a solution of imidazole (1.36 g, 20.0 mmol.) andtriethylamine (2.7 mml, 20 mmol.) in 50 ml dry methylene chloride. Atthe end of the addition, the reaction mixture was allowed to warm up toroom temperature and stirred under Ar at that temperature overnight. Thereaction mixture was then washed with 20 ml of a 10% solution ofammonium chloride, then with 20 ml of distilled water. The organic phasewas dried over magnesium sulfate and evaporated in vacuo to yieldquantitatively a white solid. Recrystallization from methylenechloride/hexanes yielded 5.60 g of (5) (yield=90% afterrecrystallization).

m.p. 214° C.; ¹ H NMR (200 MHz, CDCl₃) δ7.43 (m, 1H, NCHN), 7.3-7.4 (m,9H, 3×C₆ H₅), 7.1-7.2 (m, 6H, 3×C₆ H₅), 7.0 (m, 1H, Ph₃ CNCH═CH), 6.81(m, 1H, Ph₃ CNCH═CH); ¹³ C NMR (50 MHz, CDCl₃) δ142.3, 139.0, 129.6,128.2, 128.3, 121.6.

EXAMPLE 23

Synthesis of (S)-1-(2-(1-triphenylmethyl)-imidazolyl)-propan-2-ol(S)-(6) ##STR72##

To a solution of N-tritylimidazole (1.55 g, 5 mmol) in freshly distilledTHF (50 ml) cooled down to -78° C. and stirred under dry Ar, was added a2.5 M solution of n-butyllithium in pentane (2.4 ml, 6 mmol). Theaddition lasted for 30 min, and the deep red solution obtained wasallowed to warm up to 0° C., stirred at room temperature for 1 hour,then cooled down again to -78° C. At that temperature, (S)-propyleneoxide (0.35 g, 6 mmol) was added dropwise. After 30 min, the solutionwas allowed to warm up to 0° C. and was stirred at that temperature for12 hours until TLC indicated that the reaction did not further proceed.The solution was poured into 50 ml of saturated NH₄ Cl solution, and theresulting mixture was extracted with CH₂ Cl₂. After flash chromatography(hexane:acetone:triethylamine 78:21:1), 1.44 g of the pure product wascollected in 78% yield: m.p. 201° C.: ¹ H NMR (200 MHz, CDCl₃)δ7.20-7.40 (m, 9H, 3×C₆ H₅), 7.10-7.18 (m, 6H, 3×C₆ H₅), 6.90 (d, 1H, ³J_(H-H) =1.2; CHNCPh₃), 6.71 (d, 1H, ³ J_(H-H) =1.2; CH═CHNCPh₃), 5.83(b, 1H, OH), 3.40-3.60 (m, 1H, CHCH₃), 1.78-2.05 (ABX, 2H, ³ J_(Hb-Hx)=3.2 Hz, ³ J_(Ha-Hx) =8.5 Hz, ² J_(Ha-Hb) =16.2 Hz, CH₂), 0.81 (d, ³J_(H-H) =6.0 Hz, 3H, CH₃); ¹³ C NMR (50 Mhz, CDCl₃) δ149.2, 142.1,129.6, 127.9, 127.7, 124.7, 121.0 (NCCN), 74.6, 65.0 (CHOH), 38.1 (CH₂),22.3 (CH₃).

EXAMPLE 24

Synthesis of (S)-1-(2-imidazolyl)-propan-2-ol (S)-(7) ##STR73##

A solution of N-tritylimidazolylpropanol (S)-(6) (2.39 g, 6.51 mmol) in80 ml methanol containing 4.3 ml glacial acetic acid (5%) was refluxedfor about 12 hours. After that time, TLC indicated disappearance of thestarting materials. The mixture was concentrated in vacuo and a whiteprecipitate appeared upon addition of 50 ml of cold distilled water. Themixture was chilled, then filtered, and the white precipitate was washedwith cold distilled water (10 ml). The filtrate was then evaporatedtwice, and the residual yellow oil redissolved in 50 ml dry methanol andpassed through the weakly basic anion exchange resin (hydroxide form)IRA-68. The solution was then evaporated to yield a solid residue thatcould be recrystallized from a mixture of methanol and ethyl acetate.yield: 0.80 g, 98% of pure (S)-(7). m.p. 119-121° C.; ¹ H NMR (200 Mhz,CD₃ OD) δ6.96 (s, 2H, NCHCHN); 3.96 (m, 1H, CHCH₃); 2.4-2.65 (ABX, ³J_(Ha-Hx) =6.3 Hz, ³ J_(Hb-Hx) =6.7 Hz, ³ J_(Ha-Hb) =14.5 Hz, CH₂); 0.87(d, 3H, ³ J_(H-H) =6.3 Hz, CH₃); ¹³ C NMR (50 MHz, CD₃ OD) δ145.8 (s,CH2CNH); 121.2 (d, NCH═CHNH); 67.1 (d, CHOH); 38.4 (t, CH2); 23.1 (q,CH3); MS (CI): [M+1]⁺ 127

EXAMPLE 25

Synthesis of1-methoxy-3-methyl-imidazo-[2,1-e]-(3,4-dihydro)-oxazaphosphorine (8)##STR74##

In an NMR tube previously dried in vacuo and under Ar was introduced23.0 mg (0.20 mmol) of (S)-(2-imidazolyl)-propan-2-ol (S)-(7), then thetube was sealed with a septum and flushed with Ar. CDCl₃ (0.7 ml) wasthen introduced, followed by 127 μl (1.0 mmol) of triethylamine. Thealcohol did not dissolve, and this suspension was cooled down to 0° C.while shaking the tube. At that temperature, 18.9 μl (0.20 mmol) ofmethyl dichlorophosphite was syringed inside the tube. Upon shaking, thealcohol dissolved instantaneously, and an exothermic reaction wasnoticed. After about 1 hour, ³¹ P NMR revealed the presence of severalpeaks abound 120-140 ppm. After about 3 hours, a single product (8) wasobserved by ³¹ P NMR, as evidenced by its chemical shift at 118.8 ppm.There was no further characterization of this compound, as all effortsto isolate it have been unsuccessful and have led to the hydrolysis ofthis extremely water sensitive bicyclic structure, most likely to thecorresponding H-phosphonate.

EXAMPLE 26

Synthesis of1-ethoxy-3-methyl-imidazo-[2,1-e]-(3,4-dihydro)-oxazaphosphorine (9)##STR75##

To an NMR tube previously dried in vacuo and under Ar, was introduced18.9 mg (0.15 mmol) of (S)-(2-imidazolyl)-propan-2-ol (S)-(7). The tubewas then sealed with a septum and flushed with Ar. Then, 0.7 ml CDCl₃was introduced, followed by 105 μl (0.75 mmol) of triethylamine. Thealcohol did not dissolve, and this suspension was cooled down to 0° C.while shaking the tube. At that temperature, 17.2 μl (0.15 mmol) ofethyl dichlorophosphite was syringed into the NMR tube. Upon shaking,the alcohol dissolved instantaneously, and an exothermic reaction wasnoticed. After about 2 hours, a single produce was observed by ³¹ P NMRas evidenced by its chemical shift at 118.2 ppm. After about 1 hour, thepresence of other products around 120-140 ppm was noticed, one of which(121.1 ppm) is probably the other diastereomer. There was no furthercharacterization of this compound.

EXAMPLE 27

Synthesis of thiophosphate ethyl (S)-1-(2-imidazolyl)-prop-2-ylisopropyl ester (22) ##STR76##

In an NMR tube, to a suspension of 18.9 mg (0.15 mmol)(S)-1-(2-imidazolyl)-propan-2-ol (S)-(7) in 0.7 ml dry deuteratedchloroform and 0.21 ml triethylamine (1.5 mmol), shaken at roomtemperature under Ar was introduced with a syringe 17.2 μl (0.15 mmol)of ethyl dichlorophosphite. The reaction mixture was shaken at roomtemperature and the chiral imidazolylpropanol dissolved immediately inan exothermic process. At that point, ³¹ P NMR indicated the formationof several products, among which was one having a signal at 118.3 ppmand one having a signal at 120.5 ppm. (suspected diastereomers). Afterone hour and regular shaking of the NMR tube, ³¹ P NMR indicated onlyone peak at 118.3 ppm. At that stage, 20 μl (0.45 mmol) of isopropanolwas introduced, and the tube was shaken again. ³¹ P NMR indicated after20 min the presence of a single peak at 140.6 ppm indicating that thedisplacement of the imidazole moiety had given rise to a singlediastereomer (19). 32 mg sulfur (1mmol) was then introduced and the ³¹P-NMR was again recorded. The spectrum indicated a single peak at 64.8ppm. The product (22) was then concentrated in vacuo and purified byflash chromatography (ethyl acetate/hexanes/triethylamine 79/20/1). ¹ HNMR (200 MHz, CDCl₁ 6.96 (s, 2H, NCH═CH--N); 4.83-4.97 (m, 1H,P--O--CH--CH₃); 4.58-4.75 (dh, 1H, ³ J_(H-H) =6.2 Hz, ³ J_(H-P) =9.6 Hz,OCH (CH₃)₂); 3.96-4.12 (m, 2H, P--O--CH₂ CH₃); 2.98-3.20 (2×ABX, 2H, ²J_(Ha-H) =15.5 Hz, ³ J_(Ha-Hx) =6.23 Hz, ³ J_(Hb-Hx) =4.6 Hz, ⁴ J_(H-P)=1.5 Hz, CH₂ CHCH₃); 1.21-1.34 (m, 12H, CH(CH₃)+CH(CH₃)₂ +CH₂ CH₃) ¹³ CNMR (50 MHz, CDCl₃) 144.00 (s, N--C═N); 121.70 (s, N--C═C--N); 74.90 (d,² J_(C-P) =6.1 Hz, P--O--CH--(CH₃)CH₂); 73.80 (d, ² J_(C-P) =5.7 Hz,P--O--CH₂ --CH₃); 64.25 (d, ² J_(C-P) =5.8 Hz, P--O--CH(CH₃)₂); 35.95(s, P--O--CH(CH₃ CH₂ --); 23.40 (s, P--O--CH(CH₃)₂); 21.06 (s,P--O--CH(CH₃)CH₂ --); 15.83 (s, P--O--CH₂ --CH₃) ³¹ P NMR (81 MHz,CDCl₃) δ64.8 ppm M.S. (CI), [M+1]⁺ 293.

EXAMPLE 28

Synthesis of thiophosphate ethyl (S)-1-(2-imidazolyl)-prop-2-yl 5terbutyldimethylsilylthymidinyl ester (19) ##STR77##

To a suspension of (S)-imidazolylpropanol (S)-(7)-(0.30 mmol, 37.8 mq)in 2 ml dry dichloromethane and triethylamine (1.5 mmol, 0.21 ml) cooleddown to 0° C. and stirred under Ar, was slowly added ethyldichlorophosphite (0.30 mmol, 35 μl). The reaction mixture was thenallowed to warm up to room temperature, the solid starting materialsdissolved, and after about 2 hours, ³¹ P NMR indicated the presence of asingle peak at 118.3 ppm. At that time, the mixture was cooled down to0° C. again, and at that temperature was added a mixture of5'-tBDMS-thymidine (1) (0.30 mmol, 106 mg) in 1.5 ml of dry methylenechloride. The reaction mixture was allowed to warm up to roomtemperature again. ³¹ P NMR indicated, after 30 min, a full conversionof the peak at 118.3 ppm to a single peak at 141.2 ppm, assigned to(23). Elemental sulfur S₈ (0.9 mmol, 29 mg) was then added after about30 minutes. ³¹ P NMR indicated conversion of the previously formedproduct to a single product (24) with a peak at 66.3 ppm. Evaporation ofthe reaction mixture followed by flash chromatography (ethylacetate/triethylamine 80/20) afforded thioate (24) as a sticky solid(127 mg, 72%). ¹ H NMR (300 MHz, CD₂ Cl₂) δ 7.48 (d, 1H, ⁴ J_(H--H) =1.3Hz, C═CH); 6.94 (s, 2H, NCH═CHN); 6.25 (dd, 1H, ³ J_(H--H) =5.1 Hz, ³J_(H--H) =9.2 Hz, NCHO); 4.84-5.05 (m, 2H, POCH(CH₃)CH₂ +POCHCH₂ O);4.15-4.24 (m, 1H, SiOCH₂ CHO); 3.96-4.14 (dq, 2H, ³ J_(H--P) =9.4 Hz, ³J_(H--H) =7.0 Hz, POCH₂ CH₃); 3.76-3.92 (ABX, 2H, ² J_(Ha--Hb) =11.5 Hz,³ J_(Hb--Hx) =2.5 Hz, ³ J_(Ha--Hx) =2.4 Hz, SiOCH₂ CHO); 2.98-3.08 (dd,2H, ³ J_(H--H) =5.8 Hz, ⁴ J_(H--P) =1.1 Hz, POCH(CH₃)CH₂ --Im);1.98-2.08 (m, 2H, POCHCH₂ CHN); 1.88 (s, 3H, CH═CCH₃); 1.39 (d, 3H, ³J_(H--H) =6.2 Hz, POCH(CH₃)); 1.28 (dt, 3H, ³ J_(H--H) =7.0 Hz, ⁴J_(H--P) =0.9 Hz, POCH₂ CH₃); 0.91 (s, 9H, SiC(CH₃)₃); 0.12 (s, 6H,Si(CH₃)₂).

¹³ C NMR (121 MHz, CD₂ Cl₂, decoupled from ¹ H) δ 164.1 (s, NCOC(CH₃));151.1 (s, NCON); 144.2 (s, NC═N); 135.5 (s, NC═C(CH₃)C═O); 132.4 (s,NC═CN); 186.1 (s, 11.5 (C(CH₃)CO); 86.1 (s, SiOCH₂ CHO); 86.0 (s, NCHO);79.9 (d, ² J_(C--P) =4.4 Hz, POCH(CH₃)); 76.2 (d, ² J_(C--P) =5.7 Hz,POCH₂ CH₃); 65.0 (d, ² J_(C--P) =5.6 Hz, POCHCHO); 63.8 (s, SiOCH₂);39.4 (s, POCHCH₂ CHN); 36.6 (POCH(CH₃)CH₂); 26.1 (SiC(CH₃)₃); 21.3(POCH(CH₃)); 18.6 (SiC(CH₃)₃); 16.0 (s, POCH₂ CH₃); 12.7 (s, C═CCH₃);-5.4 (d, Si(CH₃)₂); MS (CI) (M+1)⁺. 589.

EXAMPLE 29 1,2-O-3,5-O-dicyclopentylidene-D-xylofuranose (25) ##STR78##

To a solution of trimethyl orthoformate (5 mmol, 547 μl) and p-toluenesulfonic acid (0.2 mmol, 38 mg) in dioxane (10 ml) under a nitrogenatmosphere at 0° C., was added dropwise cyclopentanone (40 mmol, 3.5ml). This solution was stirred at room temperature for 2 hours andD-xylose (2 mmol, 300 mg) was added with stirring continued overnight.Then the reaction mixture was neutralized with triethylamine.Evaporation of the solvent furnished a yellow syrupy residue. A solutionof the syrupy residue in chloroform (20 ml) was washed with water (20ml). The aqueous layer was extracted with chloroform (3×15 ml). Thecombined chloroform layers were dried (MgSO₄). After removing thesolvent, the mixture was chromatographed on a silica gel column(Hexane:Ethyl acetate=5:1) to give 340 mg white solid (60%). ¹ HNMR (200MHz, CDCl3) δ 5.99 (δ, J=3.80 Hz, 1H, H-1), 4.45 (d, J=3.81 Hz, 1H,H-2), 4.24 (d, J=2.15 Hz, 1H, H-3), 4.14-3.46 (m, 3H, H-4, 2×H-5),1.98-1.55 (m, 16H, cyclopentylidene protons); ¹³ C NMR (125 MHz, CDCl₃)δ 121.23 (C-1OCOC-2), 109.24 (C-3OCOC-5), 105.08 (C-1), 84.49 (C-2),74.25 (C-4), 71.83 (C-3), 61.50 (C-5), 39.52, 36.82, 36.19, 29.74,24.09, 23.49, 22.80, 22.37 (cyclopentylidene carbons); MS (CI) m/e: 283(M+H⁺).

EXAMPLE 30 1,2-O-cyclopentylidene-D-xylofuranose (26) ##STR79##

The 1,2-O-3,5-O-dicyclopentylidene-D-xylofuranose (25) (1 mmol, 282 mg)was dissolved in acetic acid-water (2:1) (14 ml) at room temperature.The reaction mixture was stirred for 3 hours, followed by TLC. Solventwas evaporated with high vacum, and coevaporated with methanol threetimes and dried in vacuo overnight to give 196 mg white solid (91%). ¹ HNMR (500 MHz, CDCl₃) δ 5.94 (d, J=3.91 Hz, 1H, H-1), 4.44 (d, J=3.91 Hz,H-2), 4.32 (d, J=2.44 Hz, 1H, H-3), 4.18 (m, 1H, H-4), 4.10-4.02 (m, 2H,2×H-5), 1.96-1.61 (m, 8H, cyclopentylidene protons); ¹³ C NMR (125 MHz,CDCl₃) δ 121.41 (OCO), 104.53 (C-1), 85.59 (C-2), 78.76 (C-3), 76.83(C-4), 61.07 (C-5), 36.84, 36.19 (CH₂ CCH₂), 23.47, 22.80 (CH₂ CH₂ CCH₂CH₂); MS (CI): m/e 217 (M+H⁺).

EXAMPLE 31 1,2-O-cyclopentylidene-5'-O-tosyl-D-xylofuranose (27)##STR80##

To a solution of 1,2-O-cyclopentylidene-D-xylofuranose (26) (0.81 mmol,176 mg) in dry pyridine (6 ml) under nitrogen atmosphere at 0° C.,p-toluenesulfonyl chloride (1.12 eq., 173 mg) was added. The reactionmixture was stirred at 0° C. for 3 hours. Then 5 ml water was added toquench the reaction, and the solvent was evaporated in high vacuum, andcoevaporated with toluene twice. The mixture was dissolved inchloroform, washed with water three times and dried over MgSO₄. Removalof the solvent furnished 259 mg (27) as a white solid (86%). ¹ H NMR(200 MHz, CDCl₃) δ 7.80-7.32 (AA'BB', 4H, Ph), 5.84 (d, J=3.63 Hz, 1H,H-1), 4.44 (d, J=3.7 Hz, 1H, H-2), 4.42-4.29 (m, 3H, H-3, 2×H-5),4.27-4.09 (m, 1H, H-4), 2.44 (s, 3H, CH₃), 1.89-1.61 (m, 8H,cyclopentylidene protons); ¹³ C NMR (270 MHz, CDCl₃) 145.34, 132.30,130.06, 128.08 (aromatic), 121.86 (OCO), 104.80 (C-1), 85.15 (C-2),77.73 (C-3), 74.41 (C-4), 66.21 (C-5), 37.02, 36.44 (CH₂ CCH₂), 23.54,22.98 (CH₂ CH₂ CCH₂ CH₂), 21.73 (CH₃); MS (CI): m/e 371 (M+H⁺).

EXAMPLE 32 1,2-O-dicyclopentylidene-5'-isopropylamine-D-xylofuranose(28) ##STR81##

A solution of 1,2-O-cyclopentylidene-5'-tosyl-D-xylofuranose (27) (7.1mmol, 2.64 g) in isopropylamine (15 ml) was stirred at 55° C. over nightin a pressure bottle. The solvent was removed by rotary evaporator andthe remaining yellow syrup was taken up with chloroform and washed witha saturated solution of sodium bicarbonate, and then with brine. Theorganic phase was dried over MgSO₄, the solvent was evaporated and theresidue was flash chromatographed on silica gel (ethyl acetate-3%triethylamine) to furnish 1.33 g (28) as a white solid (73%), m.p.44-45°C.; [α]_(D) ²⁰ =31.06 (C=2, CHCl₃); ¹ H NMR (500 MHz, CDCl₃) δ 8.0 (bs,NH) 5.90 (d, J=3.91 Hz, 1H, H-1), 4.38 (d, J=3.91 Hz, 1H, H-2), 4.27 (d,J=2.44 Hz, 1H, H-3), 4.20 (d, J=2.93 Hz, 1H, H-4), 3.36-2.92 (ABX, 2H,2×H-5), 2.74-2.70 (heptet, 1H, NCH), 1.95-1.63 (m, 8H, cyclopentylideneprotons), 1.04-1.03 (dd, 6H, Me₂ CH); ¹³ C NMR (500 MHz, CDCl₃) δ 121.06(OCO), 104.82 (C-1) 86.14 (C-2), 78.30 (C-3), 77.07 (C-4), 48.64 (NCH),45.90 (C-5), 36.85, 36.32 (CH₂ CCH₂), 23.51, 22.84 (CH₂ CH₂ CCH₂ CH₂),22.64 (CH₃ CHN), 22.34 (CH₃ CHN); MS (CI): m/e 258 ([M+H⁺ ], 100%;HRMS(EI) m/e calc'd for C₁₃ H₂₃ NO₄) [M⁺ ]: 257.16270, found 257.1630.

EXAMPLE 33 Chlorophosphoramidite (29) ##STR82##

In a scrupulously dry NMR tube, 9.6 μl (0.11 mmol) phosphorustrichloride was placed via syringe, followed by 0.25 ml CDCl₃. The NMRtube was cooled at 0° C., and a solution of the1,2-O-dicyclopentylidene-5'-isopropylamino-D-xylofuranose 28 (25.7 mg,0.1 mmol) and triethylamine (27.8 ul, 0.22 mmol) in CDCl₃ (0.35 ml) wereadded under a nitrogen atmosphere with shaking of the NMR tube. Anexothermic reaction was noticed. The NMR tube was then cooled to -78°C., pumped and sealed. The sealed NMR tube was heated to 40° C. and thereaction was followed by ³¹ P NMR until a single peak was found in the³¹ PNMR spectrum. The product was not isolated, and was used directly inthe following step. ¹ H NMR (500 MHz, CDCl₃) δ 5.75 (d, J=3.91 Hz, 1H,H-1), 4.51 (t, J=2.44 Hz, J=2.93 Hz, H-3), 4.37 (d, J=3.91 Hz, 1H, H-2),4.19-4.17 (m, 1H, H-4), 3.42-3.34 (heptet, 1H, NCH), 3.33-3.00 (ABX, 2H,2×H-5), 1.81-1.51 (m, 8H, cyclopentylidene protons), 1.05 (d, J=6.84 Hz,6H, Me₂ CH); ¹³ C NMR (125 MHz, CDCl₃) δ 121.48 (OCO), 104.26 (C-1),83.86, 83.83 (d, J=3.66 Hz, C-2), 73.04, 72.99 (d, J=6.41 Hz, C-3),72.35 (C-4), 50.42, 50.13 (d, J=35.72 Hz, C-5), 36.95, 36.91 (d, J=5.50Hz, NCH), 36.62, 35.91 (CH₂ CCH₂), 23.22, 22.50 (CH₂ CH₂ CCH₂ CH₂),20.89, 20.79 (d, J=12.82 Hz, CH₃ CHN), 19.64, 1960 (d, J=5.50 Hz, CH₃CHN); ³¹ P NMR (202 MHz, CDCl₃), δ 148.42.

EXAMPLE 34 5'-O-(tert-butyl dimethylsilyl)-thymidine-3'-O-Phosphoramidite (30) ##STR83##

To the same NMR tube from the example 29, a solution of5'-O-(tert-butyldimethylsilyl)-thymidine (35.6 mg, 0.1 mmol) in 0.45 mlCDCl₃ and triethylamine (14 μl, 0.11 mmol) was added slowly at 0° C.under nitrogen atmosphere. Then the NMR tube was cooled to -78° C.,pumped and sealed. The sealed NMR tube was heated to 50° C. and thereaction was followed by ³¹ P NMR until a single peak corresponding to anew product was found in the ³¹ PNMR spectrum. The solution was pouredinto flask and taken up with ethyl acetate (prewashed with a saturatedsolution of sodium bicarbonate) and washed with saturated solution ofsodium bicarbonate. The organic layer was dried over MgSO₄, the solventwas removed to furnish a white foam in a quantatitive yield. The crudeproduct was flash chromatographed on a silica gel column (hexane-ethylacetate-triethylamine=5:3:2); m.p. 68-70° C.; [α]_(D) ²⁰ =62.9° (c=0.5,CHCl₃) ¹ H NMR (500 MHz, CDCl₃) δ7.98 (bs, 1H, NH), 7.46 (d, J=1.00 Hz,1H, H-6), 6.34-6.31 (dd, J=5.86 Hz, J=8.30 Hz, 1H, H-1'), 5.88 (d,J=3.91 Hz, 1H, H-1"), 4.56-4.53 (m, 1H, H-3'), 4.41 (d, J=3.91 Hz, 1H,H-2"), 4.35 (m, 1H, H-3"), 4.17 (d, J=1.95 Hz, 1H, H-4"), 4.05 (d,J=1.95 Hz, 1H, H-4'), 3.89-3.76 (ABX, 2H, 2×H-5'), 3.45-3.39 (m, 2H,H-5", NCH), 3.03-2.99 (m, 1H, H-5"), 2.37-2.34 (m, 1H, H-2'), 2.09-2.05(m, 1H, H-2'), 1.88 (d, J=1.47 Hz, 3H, MeC═C), 1.95-1.61 (m, 8H,cyclopentylidene protons), 1.11-1.00 (m, 6H, Me₂ CH), 0.92 (s, 9H,t-BuSi), 0.09 (d, J=1.47 Hz, 6H, Me₂ Si); ¹³ C NMR (125 MHz, CDCl₃) δ163.77 (C-4), 150.35 (C-2), 135.17 (C-6), 121.42 (OCO), 110.96 (C-5),104.59 (C-1"), 86.38, 86.36 (d, J=2.75 Hz, C-4'), 84.76 (C-1'), 84.74(C-2"), 73.78, 73.63 (d, J=19.23 Hz, C-3'), 73.07, 73.05 (d, J=1.83,C-4"), 71.83, 71.80 (d, J=3.66 Hz, C-3"), 63.17 (C-5'), 50.09, 49.80 (d,J=36.63 Hz, NCH), 40.27, 40.23 (d, J=4.58 Hz, C-2'), 36.89, 36.20 (CH₂CCH₂), 36.11, 36.08 (d, J=3.21 H_(z), C-5"), 25.91 (SiCMe3), 23.44,22.76 (CH₂ CH₂ CCH₂ CH₂), 22.02, 21.95 (d, J=9.16 Hz, CH₃ CHN), 21.69,21.64 (d, J=6.41 Hz, CH₃ CHN), 18.31 (SiCMe3), 12.51 (CH3C═C), -5.41,-5.48 (d, J=9.16 Hz, Me₂ Si); ³¹ P NMR (81 MHz, CDCl₃) δ 130.14; MS(CI): m/e 642 ([M+H⁺ ], 78%); EI m/e [M⁺ ]641, HRMS (EI) m/e calc'd forC₂₈ H₄₅ N₃ O₉ PSi [M⁺ --CH₃ ]626.26625, found 626.26670, and calc'd C₂₅H₃₉ N₃ O₉ PSi [M⁺ --C₄ H₉ ]: 584.21930, found 584.2190. HRMS FAB(glycerol) m/e calcd. for C₂₉ H₄₉ N₃ O₉ PSi [MH⁺ ]642, 2975; found642.2973.

EXAMPLE 35 Protected Phosphorothioate Dinucleotide (34) ##STR84##

In a dried NMR tube, phosphoramidite 30 (15 mg, 0.0234 mmol),3'-O-(tert-butyldimethylsilyl) thymidine (10 mg, 1.2 eq.) and4,5-dicyano-2-bromo-imidazole 21 (9.17 mg, 2.0 eq.) were added. Then theNMR tube was dried in vacuum overnight. Dry acetonitile (0.6 ml) wasinjected into the NMR tube at 0° C. under nitrogen atmosphere. The soliddissolved instantly. The reaction was moved to room temperature and wasfollowed by ³¹ P NMR. Within 5 minutes, the peak corresponding to thephosphoramidite disappeared and two new peaks (143.76 ppm, 142.55ppm=6:1) were formed. The clean ³¹ P NMR spectrum suggested use of thisproduct directly in the following sulphurization without isolation. Tothis solution, Beaucage's reagent (5.6 mg, 1.2 eq.) in 140 ulacetonitrile (0.2M) was added. Instantaneously the ³¹ P NMR showedanother two peaks (68.43 ppm, 68.23 ppm=1:6), while the peakscorresponding to the starting material disappeared. The solution in NMRtube was transfered to a flask. Then the mixture was redisolved in ethylacetate, washed with saturated sodium bicarbonate and water, and driedover MgSO₄. Removal of the solvent gave 22 mg of white solid 34 (91%).The product was then purified by chromatography on a silica gel column(ethyl acetate:methanol=95:5). ¹ H NMR (500 MHz, DMSO-d6) δ 7.45 (s, 1H,³ H-6), 7.42 (s, 1H, ⁵ H-6), 6.19-6.15 (m, 2H, ⁵ H-1', ³ H-1'), 5.86,5.85 (d, J=3.91 Hz, 1H, H-1"), 5.05-5.04 (m, 1H, ⁵ H-3'), 4.78-4.77 (m,1H, H-3"), 4.60, 4.59 (d, J=3.42 Hz, 1H, H-2"), 4.38-4.36 ((m, 1H,H-4"), 4.26-4.13 (m, 4H, ³ H-3', 2×³ H-5', ⁵ H-4'), 3.93, 3.91 (m, 1H, ³H-4'), 3.81-3.73 (m, 2H, 2×⁵ H-5'), 2.66-2.52 (m, 3H, NCH, 2×H-5"),2.43-2.40 (m, 1H, ⁵ H-2'), 2.31-2.22 (m, 2H, 2×³ H-2'), 2.09-2.07 (m,1H, ⁵ H-2'), 1.78 (s, 3H, ³ CH₃ C═C), 1.76 (s, 3H, ⁵ CH₃ C═C), 1.62-1.56(m, 8H, cyclopentylidene protons), 0.94-0.89 (m, 6H, Me₂ CHN), 0.86 (s,9H, ³ t-BuSi), 0.85 (s, 9H, ⁵ t-BuSi), 0.069 (s, 6H, Me₂ Si), 0.065 (s,6H, Me₂ Si); ¹³ CNMR (125 MHz, CDCl₃) δ 163.97, 163.66 (⁵ C-4, ³ C-4),150.39, 150.04 (⁵ C-2, ³ C-2), 135.58, 134.49 (⁵ C-6, ³ C-6), 121.77(OCO), 111.26, 111.04 (⁵ C-5, ³ C-5), 104.00 (C-1"), 85.59, 85.56 (d,J=6.41 Hz, ⁵ C-4'), 85.48 (³ C-1'), 84.69 84.61, 84.39 (⁵ C-1', ³ C-4'),83.47 (C-2"), 80.85, 80.64 (d, C-3"), 80.69, 80.66 (d, ⁵ C-3'), 79.03,78.97 (d, ³ C-3'), 71.49 (C-4"), 67.66, 67.52 (d, J=4.58 Hz, ³ C-5'),63.47 (⁵ C-5'), 48.93 (NCH), 45.23 (C-5"), 40.37 (³ C-2'), 39.14, 39.09(d, J=6.41 Hz, ⁵ C-2'), 37.06, 36.16 (CH₂ CCH₂), 25.88, 25.60 (⁵ SiCMe₃,³ SiCMe₃), 23.52, 22.85 (CH₂ CH₂ CCH₂ CH₂), 22.65 22.39 (NCHMe₂), 18.28,17.81 (⁵ SiCMe₃, 3SiCMe₃), 12.51, 12.46 (⁵ C═CCH₃, ³ C═CCH₃), -4.66,-4.83, -5.40, -5.46 (⁵ SiMe2, ³ SiMe₂); ³¹ P NMR (202 MHz, CDCl₃) δ68.43, 68.23 (1:6) MS (FAB): m/e 1030 (M+H⁺).

(II). 4,5-dicyano-2-bromo-imidazole 21 (7.6 mg, 2.5 eq.),phosphoramidite 30 (10 mg, 0.0156 mmol) and3'-O-(tert-butyldimethylsilyl)thymidine (6.7 mg, 1.2 eq.) were added toa dried NMR tube. The the NMR tube was dried under vacuum overnight, anddry CDCl₃ (0.5 ml) was injected into the NMR tube at 0° C. under anargon atmosphere. The reaction was followed by ³¹ P NMR until thereaction went to completion. The ³¹ P NMR spectrum showed that the peakcorresponding to the phosphoramidite at 130 ppm had disappeared, andthat two new peaks appeared at 142.634, 141.880 ppm in a ratio of 40:1.MS(FAB) [M+H+]: calc'd for C₄₅ H₇₆ N₅ PO₁₄ Si₂ phosphite triester 998,found 998.4.

Beaucage's reagent (3.8 mg, 1.2 eq.) was added directly to the solution.The ³¹ PNMR (202 MHz, CDCl₃, 0° C.) instantaeously showed another twopeaks at 67.831 and 67.514 ppm in the same ratio, while the peakscorresponding to the phosphite triester disappeared. The solution in theNMR tube was transfered to a flask, the solvent was evaporated, and theproduct was then purified by chromatography on a silica gel column(ethyl acetate:hexane:triethylamine=60:35:5) to give only one isomer. ³¹PNMR (202 MHz, CDCl₃, room temperature) δ 68.291.

(III) Virtually identical results were obtained when the reaction wascarried out at -15° C. for 7 hours in CDCl₃, except that the ratio ofisomers was .sup.˜ 68:1. MS FAB (nitrobenzyl alcohol): m/e [MH⁺ ]1030HRMS FAB CsI m/e calcd. for C₄₅ H₇₇ N₅ O₁₄ PSSi₂ [MH⁺ ], 1030.4464;found 1030.4460.

EXAMPLE 36 Phosphorothioate Dinucleotide (35) ##STR85##

Protected phosphorothioate dinucleotide 34 (14 mg, 0.0136 mmol) fromExample 35 was dissolved in 1 ml 70% TFA--H₂ O at 0° C. with stirring,and then the reaction was allowed to proceed at room temperature. Thereaction was followed by TLC until the spot corresponding to thestarting material disappeared. Evaporation of the solvent andcoevaporation with methanol three times furnished a white solid. Thecrude product was purified with preparative TLC plate (0.5 mm) (CH₂ Cl₂:MeOH=5:1) to give 7.2 mg of a white solid (35) (94%); ¹ H NMR (500 MHz,CD₃ OD)δ67.87 (s, 1H, ³ H-6), 7.85 (s, 1H, ⁵ H-6), 6.36-6.33 (dd, J=6.35Hz, J=7.81 Hz, 1H, ⁵ H-1'), 6.30-6.27 (dd, J=6.35 Hz, J=7.33 Hz ³ H-1'),5.06-5.03 (m, 1H, ⁵ H-3') 4.53-5.52 (m, 1H, ³ H-3'), 4.21-4.06 (m, 4H, ⁵H-4', 2×³ H-5', ³ H-4'), 3.84-3.80 (m, 2H, 2×⁵ H-5'), 2.50-2.46 (m, 1H,⁵ H-2'), 2.31-2.24 (m, 2H, 2×³ H-2'), 2.21-2.17 (m, 1H, ⁵ H-2'), 1.96(s,3H, ³ CH₃ C═C), 1.87(s, 3H, ⁵ CH₃ C═C); ³¹ P NMR (202 MHz, CD₃OD)δ58.64:58.57 (6:1) MS (FAB):m/e 563 (M+H⁺).

EXAMPLE 37 1,2-O-3,5-O-dicyclopentylidene-L-xylofuranose (36) ##STR86##

To a solution of trimethyl orthoformate (68 mmol, 7.5 ml) and p-toluenesulfonic acid (2 mmol, 380 mg) in dioxane (45 ml) under a nitrogenatmosphere at 0° C., was added dropwise cyclopentanone (500 mmol, 45ml). This solution was stirred at room temperature for 2 hours andL-xylose (20 mmol, 3.0 g) was added with stirring continued overnight.Then the reaction mixture was neutralized with triethylamine.Evaporation of the solvent furnished a yellow syrupy residue. A solutionof the syrupy residue in chloroform (50 ml) was washed with water (50ml). The aqueous layer was extracted with chloroform (3×20 ml), and thecombined chloroform layers were dried over (MgSO₄). After removing thesolvent, the mixture was chromatographed on a silica gel column(hexane:ethyl acetate=5:1) to give 3.5 g white solid 36 (62%). ¹ HNMR(500 MHz, CDCl₃)δ5.96 (d, J=3.91 Hz, 1H, H-1), 4.42 (d, J=3.91 Hz, 1H,H-2), 4.21 (d, J=1.95 Hz, 1H, H-3), 4.09-3.98 (m, 3H, H-4, 2×H-5),1.97-1.57 (m, 16H, cyclopentylidene protons); ¹³ C NMR (125 MHz,CDCl₃)δ121.20 (C-1OCOC-2), 109.20 (C-3OCOC-5), 105.05 (C-1), 84.46(C-2), 74.22 (C-4), 71.80 (C-3), 61.47 (C-5), 39.49, 36.80, 36.16,29.71, 24.07, 23.47, 22.77, 22.34 (cyclopentylidene carbons); MS (CI)m/e:283(M+H⁺).

EXAMPLE 38 1,2-O-cyclopentylidene-L-xylofuranose (37) ##STR87##

1,2-O-3,5-O-dicyclopentylidene-L-xylofuranose (36) (10 mmol, 2.82 g) wasdissolved in acetic acid-water (2:1) (60 ml) at room temperature. Thereaction mixture was stirred for 7 hours. The reaction was followed byTLC. The solvent was evaporated with high vacum, coevaporated withmethanol three times, and dried in vacuo overnight to give 2.16 g whitesolid 37 (100%). ¹ H NMR (500 MHz, CDCl₃)δ5.92 (d, J=3.91 Hz, 1H, H-1),4.42(d, J=3.91 Hz, 1H, H-2), 4.30 (d, J=2.93 Hz, 1H, H-3), 4.17-4.14 (m,1H, H-4), 4.07-3.97 (m, 2H, 2×H-5), 1.96-1.60 (m, 8H, cyclopentylideneprotons); ¹³ C NMR (125 MHz, CDCl₃)δ121.42 (OCO), 104.52 (C-1), 85.56(C-2), 78.81 (C-3), 76.77 (C-4), 61.01 (C-5), 36.84, 36.19 (CH₂ CCH₂),23.47, 22.79 (CH₂ CH₂ CCH₂ CH₂); MS (CI):m/e 217 (M+H⁺).

EXAMPLE 39 1,2-O-cyclopentylidene-5'-O-tosyl-L-xylofuranose (38)##STR88##

To a solution of 1,2-O-cyclopentylidene-L-xylofuranose (37) (9.4 mmol,2.03 g) in dry pyridine (25 ml) under nitrogen atmosphere at 0° C.,p-toluenesulfonyl chloride (1.2 eq., 2.15 g) was added. The reactionmixture was stirred at 0° C. for 12 hours. Then 10 ml water was added toquench the reaction, and the solvent evaporated in high vacum, andcoevaporated with toluene twice. The mixture was dissolved inchloroform, washed with water three times and dried over MgSO₄. Removalof the solvent furnished 2.96 g of 38 as a white solid (85%). ¹ H NMR(500 MHz, CDCl₃)δ7.78-7.32 (AA'BB', 4H, Ph), 5.83 (d, J=3.42 Hz, 1H,H-1), 4.42 (d, J=3.91 Hz, 1H, H-2), 4.42-4.24 (m, 3H, H-3, 2×H-5),4.16-4.08 (m, 1H, H-4), 2.42 (s, 3H, CH₃), 1.90-1.63 (m, 8H,cyclopentylidene protons); ¹³ C NMR (270 MHz, CDCl₃)δ145.25, 130.26,130.09, 130.01, 129.96 (aromatic), 121.71 (OCO), 104.69 (C-1), 85.04(C-2), 77.67 (C-4), 74.25 (C-3), 66.39 (C-5), 36.89, 36.30(CH₂ CCH₂),23.42, 22.85 (CH₂ CH₂ CCH₂ CH₂), 21.63 (CH₃); MS (CI):m/e 371 (M+H⁺).

EXAMPLE 40 1,2-O-dicyclopentylidene-5'-isopropylamine-L-xylofuranose(39) ##STR89##

A solution of 1,2-O-cyclopentylidene-5'-tosyl-L-xylofuranose (38) (7.0mmol, 2.6 g) in isopropylamine (15 ml) was stirred at 55° C. overnightin a pressure bottle. The solvent was removed by rotary evaporator andthe remaining yellow syrup was taken up with chloroform and washed witha saturated solution of sodium bicarbonate and with brine. The organicphase was dried over MgSO₄, the solvent was evaporated and the residuewas flash chromatographed on silica gel (ethyl acetate-3% triethylamine)to furnish 1.30 g white solid 39 (72%). m.p. 39-41° C.; [α]_(D) ²⁰=-31.37 (c=2, CHCl₃); ¹ H NMR (500 MHz, CDCl₃)δ8.0 (bs, NH), 5.88 (d,J=3.91 Hz, 1H, H-1), 4.37 (d, J=3.91 Hz, 1H, H-2), 4.25 (d, J=2.93 Hz,1H, H-3), 4.19 (m, 1H, H-4), 3.34-2.91 (ABX, 2H, 2×H-5), 2.73-2.71(heptet, 1H, NCH), 1.92-1.61 (m, 8H, cyclopentylidene protons),1.03-1.01(dd, J=2.44 Hz, J=6.35 Hz, 6H, Me₂ CH); ¹³ C NMR (500 MHz,CDCl₃)δ121.00 (OCO), 104.75 (C-1), 86.06 (C-2), 78.20 (C-3), 77.00(C-4),48.62(NCH), 45.82(C-5), 36.78, 36.25(CH₂ CCH₂), 23.46, 22.78(CH₂ CH₂CCH₂ CH₂), 22.57(CH₃ CHN), 22.27(CH₃ CHN); MS (CI):m/e 258([M+H⁺ ];100%); HRMS(EI) m/e calc'd for C₁₃ H₂₃ NO₄ [M⁺ ]:257.16270, found257.16250.

EXAMPLE 41 Chlorophosphoramidite (40) ##STR90##

In a scrupulously dry NMR tube, 9.6 μl (0.11 mmol) phosphorustrichloride was added via syringe, then 0.25 ml CDCl₃ was similarlyadded. This NMR tube was cooled to 0° C., and a solution of the1,2-0-dicyclopentylidene-5'-isopropylamine-L-xylofuranose (17) (25.7 mg,0.1 mmol) and triethylamine (27.8 ul, 0.22 mmol) in CDCl₃ (0.35 ml) wasadded under a nitrogen atmosphere with shaking of the NMR tube. Anexothermic reaction was noticed. The NMR tube was then cooled to -78°C., pumped and sealed. The sealed NMR tube was heated to 40° C. and thereaction was followed by ³¹ PNMR until a single peak was found in the ³¹PNMR spectrum. The product was not isolated, and was directly used inthe following step. ³¹ P NMR (300 MHz, CDCl3)δ148.75

EXAMPLE 42 5'-O-(tert-butyl dimethylsilyl)-thymidine-3'-O-Phosphoramidite (41) ##STR91##

To the same NMR tube from previous Example 41, a solution of5'-O-(tert-butyl dimethyl silyl)-thymidine (35.6 mg, 0.1 mmol) in 0.45ml CDCl₃ and triethylamine (14 μl, 0.11 mmol) was added slowly at 0° C.under a nitrogen atmosphere. Then the NMR tube was cooled to -78° C.,pumped and sealed. The sealed NMR tube was heated to 50° C. and thereaction was followed by ³¹ PNMR until a single peak corresponding to anew product was found in the ³¹ PNMR spectrum. The solution was pouredinto a flask, and taken up with ethyl acetate (prewashed with asaturated solution of sodium bicarbonate) and washed with saturatedsolution of sodium bicarbonate. The organic layer was dried over MgSO₄,and the solvent was removed to furnish a white foam in a quantatitiveyield. The crude pruduct was flash chromatographyed on a silica gelcolumn (hexane-ethyl acetate-triethylamine=5:3:2) to furnish whitecrystals (41); m.p. 99-101° C.; [α]_(D) ²⁰ -72.0° (c=0.5,CDCl₃) ¹ H NMR(500 MHz, CDCl₃)δ8.77 (bs, 1H, NH), 7.46(s, 1H, H-6), 6.33-6.30(dd,J=5.86 Hz, J=7.81 Hz, 1H, H-1'), 5.88(d, J=3.91 Hz, 1H, H-1"),4.56-4.53(m, 1H, H-3'), 4.43(d, J=3.42 Hz, 1H, H-2"), 4.35(m, 1H, H-3"),4.18 (d, J=1.95 Hz, 1H, H-4"), 4.05 (m, 1H, H-4'), 3.90-3.76 (ABX, 2H,2×H-5'), 3.45-3.42(m, 2H, H-5", NCH), 3.03-2.99 (m, 1H, H-5"), 2.38-2.35(m, 1H, H-2'), 2.12-2.06(m, 1H, H-2'), 1.89(s, 3H, (MeC═C), 1.96-1.62(m,8H, cyclopentylidene protons), 1.11-1.08 (m, 6H, Me₂ CH), 0.90(s, 9H,t-BuSi), 0.09, (d, J=1.95 Hz, 6H, Me₂ Si); ¹³ C NMR (125 MHz,CDCl₃)δ163.74 (C-4), 150.31(C-2), 135.18(C-6), 121.44(OCO), 110.93(C-5),104.59(C-1"), 86.61, 86.57(d, J=5.49 Hz, C-4'), 84.75, 84.73(d, J=2.75Hz, C-2'), 84.70(C-2"), 73.07, 73.05(d, J=1.83 Hz, C-3'), 73.03,72.89(d, J=17.4, C-4"), 71.82, 71.78(d, J=4.58 Hz, C-3"), 62.98(C-5'),50.05, 49.76(d, J=36.63 Hz, NCH), 39.94, 39.92(d, J=2.75 Hz, C-2'),36.86, 36.19(CH₂ CCH₂), 36.11, 36.08(d, J=3.06 H_(z), C-5"),25.92(SiCMe3), 23.45, 22.76(CH₂ CH₂ CCH₂ CH₂), 21.99, 21.91(d, J=9.16Hz, CH₃ CHN), 21.69, 21.64(d, J=6.41 Hz, CH₃ CHN), 18.35(SiCMe3),12.52(CH3C═C), -5.39, -5.45 (d, J=9.16 Hz, Me₂ Si); ³¹ P NMR (81 MHz,CDCl₃)δ129.34; MS (CI):m/e 642(M+H⁺). EI:m/e 641[M+]. MS FAB(nitrobenzyl alchol):m/e [MH⁺ ]642, HRMS FAB (glcerol) m/e calcd. forC₂₉ H₄₉ N₃ O₉ PSi [MH⁺ ]642.2975; found 642.2973.

EXAMPLE 43 Protected phosphorothiaoate dinucleotide (42) ##STR92## I: Ina dried NMR tube, phosophoramidite 41 (15 mg, 0.0234 mmol),3'-O-(tert-butyldimethylsilyl) thymidine (10 mg, 1.2 eq.) and4,5-dicyano-2-bromo-imidazole 21 (9.17 mg, 2.0 eq.) were added. Then theNMR tube was dried in vacuum overnight. Dry acetonitile (0.6 ml) wasinjected into the NMR tube at room temperature under a nitrogenatmosphere. The solid dissolved instantly. The reaction was followed by³¹ PNMR. Within 5 minutes, the peak corresponding to the phosphoramiditedisappeared. To this solution, Beaucage's reagent (5.6 mg, 1.2 eq.) in140 μl acetonitrile (0.2M) was added. The peaks corresponding to thestarting material disappeared. The solution in NMR tube was transferredto a flask, and the solvent evaporated. Then the mixture was redisolvedin ethyl acetate, washed with saturated sodium bicarbonate and water,and dried over MgSO₄. After removal of the solvent, the product waspurified by chromatography on a silica gel column (ethylacetate:methanol=95:5) to furnish 42 in a diastereomeric ratio of7:1(69.12, 68.91 ppm). ¹ H NMR (500 MHz, CDCl₃)δ7.46(s, 1H, ³ H-6),7.27(s, 1H, ⁵ H-6), 6.34-6.10(m, 2H, ⁵ H-1', ³ H-1'), 5.86, 5.87(d,J=3.42 Hz, 1H, 1H, H-1"), 5.17-5.14(m, 1H, ⁵ H-3'), 4.83-4.81(d, J=10.25Hz,1H, H-3"), 4.56, 4.55(d, J=3.91 Hz, 1H, H-2"), 4.38(m, 2H, ³ H-3',H-4"), 4.25-4.21(m, 3H, 2×³ H-5', ⁵ H-4'), 3..98(m, 1H, ³ H-4'),3.91-3.85(m, 2H, 2×⁵ H-5'), 2.85, 2.84(d, J=6.35 Hz, 1H, 2×H-5"),2.81(m, 1H, NCH), 2.52-2.47(dd, 2H, ⁵ H-2'), 2.25-2.23(m, 2H, 2×³ H-2'),2.08-2.02(m, 1H, ⁵ H-2'), 1.91(s,3H, ⁵ CH₃ C═C), 1.89(s, 3H, ³ CH₃ C═C),1.92-1.65(m, 8H, cyclopentylidene protons), 1.05, 1.04(d, J=5.86 Hz, 6H,Me₂ CHN), 0.90(s, 9H, ³ t-BuSi), 0.86(s, 9H, ⁵ t-BuSi), 0.11(s, 6H, Me₂Si), 0.05(s, 6H, Me₂ Si); ¹³ CNMR (125 MHz, CDCl₃)δ163.80, 163.59(⁵ C-4,³ C-4), 150.33, 150.15(⁵ C-2, ³ C-2), 136.13, 134.64(⁵ C-6, ³ C-6),122.03(OCO), 111.43, 111.11(⁵ C-5, ³ C-5), 104.28(C-1"), 86.29(³ C-1'),85.95, 85.90(d, J=6.41 Hz, ⁵ C-4'), 84.80, 84.82, 84.69 (⁵ C-1', ³C-4'), 83.59(C-2"), 80.85, 80.81(d, J=4.58 Hz, C-3"), 80.69, 80.66(d,J=4.58 Hz, ⁵ C-3'), 79.03, 78.97(d, J=8.24 Hz, ³ C-3'), 71.30(C-4"),67.38, 67.34(d, J=5.50 Hz, ³ C-5'), 63.37(⁵ C-5'), 48.91(NCH),45.18(C-5"), 40.35(³ C-2'), 39.12, 39.09(d, J=3.66 Hz, ⁵ C-2'), 37.14,36.23(CH₂ CCH₂), 25.88, 25.65(⁵ SiCMe₃, ³ SiCMe₃), 23.55, 22.89(CH₂ CH₂CCH₂ CH₂), 22.80, 22.55(NCHMe₂), 18.28, 17.86(⁵ SiCMe₃, 3SiCMe₃), 12.52,12.49(⁵ C═CCH₃, ³ C═CCH₃), -4.66, -4.83, -5.40, -5.46(⁵ SiMe2, ³ SiMe₂);³¹ P NMR (121 MHz, CDCl₃)δ69.12, 68.91 (7:1); MS (FAB):m/e (M+H⁺).EXAMPLE 44 Phosphorothioate dinucleotide (43) ##STR93##

The protected phosphorthioate dinucleotide 42 obtained from example 43-I(15 mg, 0.014 mmol) was dissolved in 1 ml 70% TFA-H₂ O at 0° C. withstirring and, the reaction was allowed to proceed at room temperature.The reaction was followed by TLC until the spot corresponding to thestarting material disappeared. Evaporation of the solvent andcoevaporation with methanol three times furnished a white solid. Thecrude product was purified with preparative TLC plate (0.5 mm) (CH₂ Cl₂:MeOH=5:1) to give 43 as a white solid: ¹ H NMR (500 MHz, CD₃ OD)δ7.91(s, 1H, ³ H-6), 7.86(s, 1H, ⁵ H-6), 6.36-6.33(dd, J=6.35 Hz, J=7.81 Hz,1H, ⁵ H-1'), 6.29-6.26(dd, J=5.86 Hz, J=7.81 Hz ³ H-1'), 5.08-5.05(m,1H, ⁵ H-3') 4.52-3.51(m, 1H, ³ H-3'),4.21(m, 1H, ⁵ H-4'), 4.14-4.11(m,2H, 2×³ H-5'), 4.04 (m, 1H, ³ H-4'), 3.86-3.79(m, 2H, 2×⁵ H-5'),2.48-2.44(m, 1H, ³ H-2'), 2.31-2.24(m, 2H, 2×⁵ H-2'), 2.23-2.16(m, 1H, ³H-2'), 1.97(s, 3H, ³ CH₃ C═C), 1.87(s, 3H, ⁵ CH₃ C═C); ³¹ P NMR (121MHz, CDCl₃)δ59.14:59.08(1:7); MS (FAB):m/e (M+H⁺)

EXAMPLE 45 Synthesis of γ-aminoalcohol 44 (Scheme 1)

The synthesis of amino-alcohol 44 is shown in Scheme 1 below: ##STR94##

Starting from L-mannonic-γlactone 51, both diols are protected byconversion to their acetonides according to standard procedures, usingacetone with para-toluenesulfonic acid as a catalyst, to yieldbis-acetonide lactone 52.

The bis-acetonide is then reacted in presence of samarium iodide in amixture of THF and ethylene glycol at room temperature according to theprocedure described by Christian Girard, Ph.D. Thesis, Universite deMontreal, 1995. This reaction proceeds with high yield and highregioselectivity and affects the acetonide located on the α-position tothe ester, yielding β-hydroxyester 53.

The next step is a classical acid-catalyzed deprotection of theacetonide to give triol product 54. The diol function of the triol isthen cleaved by sodium periodate on alumina in methanol. Theintermediate aldehyde undergoes a reductive amination in presence ofisopropylamine and sodium cyanoborohydride to yield γ-aminoalcohol 55,which is isolated and stabilized as the hydrochloride salt. See RobertHambalek, Ph.D. Thesis, McGill University, 1992.

γ-aminoalcohol 55 is then employed as a chiral precursor to thestereocontrolled synthesis of a P-chiral phosphorothioate dimer that canbe deprotected by a base-catalyzed β-elimination. An example of thistype of elimination is found in Takahata et al., J. Org. Chem. 1995, 60,5628-5633.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all equivalent variations as fall within the truespirit and scope of the invention.

It is intended that each of the patents, publications, and otherpublished documents mentioned or referred to in this specification beherein incorporated by reference in their entirety.

What is claimed is:
 1. A method for the preparation of aninternucleotide linkage comprising the steps of:selecting a nucleosidephosphoramidite; reacting said nucleoside phosphoramidite with anucleoside in the presence of a catalyst, said nucleoside having a free5'-hydroxyl group; thereby forming said internucleotide linkage; saidcatalyst having the formula: ##STR95## wherein: R¹² and R¹³ areindependently hydrogen, cyano, nitro, alkyl having from one to 10carbons, substituted alkyl having from one to 10 carbons, an estergroup, or R¹² and R¹³ together with the carbon atoms to which they areattached, form a substituted phenyl ring where said substituents areelectron withdrawing; and R¹⁴ is hydrogen, halogen, cyano, nitro, thio,alkyl having from one to 10 carbons, substituted alkyl having from oneto 10 carbons, norbornyl, substituted norbornyl, aryl, substituted arylwherein said substituents are electron withdrawing, or has the formula:##STR96## wherein L is a protecting group; provided that R₁₂, R₁₃ andR₁₄ are not simultaneously H; further provided that where one R₁₂, R₁₃or R₁₄ is nitro, then at least one of R₁₂, R₁₃ and R₁₄ cannot be H; andfurther provided that at least one R₁₂, R₁₃ and R₁₄ is electronwithdrawing.
 2. The method of claim 1 wherein said internucleotidelinkage is a phosphite linkage.
 3. The method of claim 1 furthercomprising the step of oxidizing said internucleotide linkage to form aphosphodiester linkage, a phosphorothioate linkage, or aphosphorodithioate linkage.
 4. The method of claim 1 wherein saidcatalyst has the structure: ##STR97## wherein: R¹² and R¹³ areindependently hydrogen, cyano, nitro, alkyl having from one to 10carbons, substituted alkyl having from one to 10 carbons, where saidsubstituents are electron withdrawing; andR¹⁴ is hydrogen, halogen,cyano, nitro, thio, alkyl having from one to 10 carbons, substitutedalkyl having from one to 10 carbons, aryl, or substituted aryl whereinsaid substituents are electron withdrawing.
 5. The method of claim 1wherein R¹², R¹³ and R¹⁴ are independently hydrogen, or cyano.
 6. Themethod of claim 1 wherein R¹² and R¹³ are each cyano, and R¹⁴ ishydrogen.
 7. The method of claim 1 wherein R¹² and R¹³ are each cyano,and R¹⁴ is halogen.
 8. The method of claim 7 wherein R¹⁴ is bromine. 9.The method of claim 2 wherein said catalyst has the structure: ##STR98##wherein: R¹² and R¹³ are independently hydrogen, cyano, nitro, alkylhaving from one to 10 carbons, substituted alkyl having from one to 10carbons, where said substituents are electron withdrawing; andR¹⁴ ishydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10carbons, substituted alkyl having from one to 10 carbons, aryl, orsubstituted aryl wherein said substituents are electron withdrawing. 10.The method of claim 3 wherein R¹², R¹³ and R¹⁴ are independentlyhydrogen, or cyano.
 11. The method of claim 10 wherein R¹² and R¹³ areeach cyano, and R¹⁴ is hydrogen.
 12. The method of claim 10 wherein R¹²and R¹³ are each cyano, and R¹⁴ is halogen.
 13. The method of claim 12wherein R¹⁴ is bromine.
 14. A method for the preparation of an activatednucleoside phosphoramidite comprising the steps of:selecting anucleoside having a free hydroxyl group; reacting said hydroxyl group ofsaid nucleoside with a halogenated trivalent phosphorus compound in thepresence of a catalyst; thereby forming said activated nucleosidephosphoramidite; said catalyst having the formula: ##STR99## wherein:R¹² and R¹³ are independently hydrogen, halogen, cyano, alkyl havingfrom one to 10 carbons, substituted alkyl having from one to 10 carbons,where said substituents are electron withdrawing; and R¹⁴ is hydrogen,halogen, cyano, thio, alkyl having from one to 10 carbons, substitutedalkyl having from one to 10 carbons, aryl, or substituted aryl whereinsaid substituents are electron withdrawing.
 15. The method of claim 14wherein said free hydroxyl group is a 3' or 5' hydroxyl group.
 16. Themethod of claim 14 wherein said halogenated trivalent phosphoruscompound is a chlorinated trivalent phosphorus compound.
 17. A methodfor the preparation of an activated nucleoside phosphoramiditecomprising the steps of:selecting a nucleoside having a free hydroxylgroup; reacting said hydroxyl group of said nucleoside with a trivalentphosphorus compound in the presence of a catalyst, where said trivalentphosphorus compound includes at least one hetero atom selected fromnitrogen and oxygen covalently bonded to a phosphorus atom; therebyforming said activated nucleoside phsophoramidite; said catalyst havingthe formula: ##STR100## wherein: R¹² and R¹³ are independently hydrogen,halogen, cyano, alkyl having from one to 10 carbons, substituted alkylhaving from one to 10 carbons, where said substituents are electronwithdrawing; and R¹⁴ is hydrogen, halogen, cyano, thio, alkyl havingfrom one to 10 carbons, substituted alkyl having from one to 10 carbons,aryl, or substituted aryl wherein said substituents are electronwithdrawing.
 18. The method of claim 17 wherein said free hydroxyl groupis a 3' hydroxyl group or a 5' hydroxyl group.
 19. The method of claim17 wherein said trivalent phosphorus compound is a halogenated trivalentphosphorus compound.
 20. The method of claim 17 said trivalentphosphorus compound is a chlorinated phosphorus compound.
 21. The methodof claim 17 wherein said trivalent phosphorus compound includes at leastone nitrogen hetero atom covalently bonded to a phosphorus atom.
 22. Themethod of claim 21 wherein said nitrogen atom is further substitutedwith an alkyl or aralkyl group having up to 15 carbon atoms.
 23. Themethod of claim 22 wherein said nitrogen atom is further substitutedwith an alkyl group.
 24. The method of claim 23 wherein said alkyl groupis isopropyl or t-butyl.
 25. The method of claim 17 wherein saidtrivalent phosphorus compound includes one nitrogen and one oxygen atomcovalently bonded to a phosphorus atom.
 26. The method of claim 25wherein said trivalent phosphorus compound further includes a halogenatom covalently bonded to said phosphorus atom.
 27. The method of claim25 wherein each of said nitrogen atom and said oxygen atoms are furthersubstituted with at least one carbon atom containing species.